Treating cancer using a blautia strain

ABSTRACT

Provided herein are methods and compositions related to  Blautia  Strain A useful as therapeutic agents.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Applications having Ser. Nos. 62/551,554, filed Aug. 29, 2017,62/573,406, filed Oct. 17, 2017, and 62/696,429, filed Jul. 11, 2018,the contents of each of which are hereby incorporated herein byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Nov. 19, 2018, is namedETB-008_01_SL.txt and is 799 bytes in size.

SUMMARY

In certain aspects, provided herein are methods and compositions relatedto the treatment of a cancer in a subject (e.g., a human subject)comprising administering a bacterial composition comprising BlautiaStrain A. In some embodiments, the Blautia Strain A is Blautiamassiliensis Strain A (ATCC Deposit Number PTA-125134). In someembodiments, the Blautia Strain A is a strain comprising at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identity(e.g., at least 99.5% sequence identity, at least 99.6% sequenceidentity, at least 99.7% sequence identity, at least 99.8% sequenceidentity, at least 99.9% sequence identity) to the nucleotide sequenceof the Blautia Strain A. In some embodiments, provided here arebioreactors comprising Blautia Strain A disclosed herein. In someembodiments, the administration of the bacterial composition induces animmune response against a tumor in the subject. In some embodiments, theadministration of the bacterial composition treats the cancer in thesubject. In some embodiments, the administration augments a tumormicroenvironment in the subject. In some embodiments, the cancer is acolorectal carcinoma.

In certain embodiments, provided herein are methods of treating asubject who has cancer, comprising administering to the subject abacterial composition comprising Blautia Strain A (e.g., a killedbacterium, a live bacterium and/or an attenuated bacterium). In someembodiments, the Blautia Strain A is Blautia massiliensis Strain A (ATCCDeposit Number PTA-125134). In some embodiments, the Blautia Strain A isa strain comprising at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity (e.g., at least 99.5% sequenceidentity, at least 99.6% sequence identity, at least 99.7% sequenceidentity, at least 99.8% sequence identity, at least 99.9% sequenceidentity) to the nucleotide sequence of the Blautia Strain A. In someembodiments, at least 50%, 60%, 70%, 80%, 85%, 90%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the bacteria in the bacterialcomposition are Blautia Strain A. In some embodiments, all orsubstantially all of the bacteria in the bacterial formulation areBlautia Strain A. In some embodiments, the bacterial formulationcomprises at least 1×10⁵, 5×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶,6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 1×10⁸,2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸ or 1×10⁹ colonyforming units of Blautia Strain A.

In certain embodiments, provided herein are bacterial compositionscomprising Blautia Strain A (e.g., a killed bacterium, a live bacteriumand/or an attenuated bacterium). In some embodiments, at least 50%, 60%,70%, 80%, 85%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%of the bacteria in the bacterial composition are Blautia Strain A. Insome embodiments, the Blautia Strain A is Blautia massiliensis Strain A(ATCC Deposit Number PTA-125134). In some embodiments, the BlautiaStrain A is a strain comprising at least 99% sequence identity (e.g., atleast 99.5% sequence identity, at least 99.6% sequence identity, atleast 99.7% sequence identity, at least 99.8% sequence identity, atleast 99.9% sequence identity) to the nucleotide sequence of the BlautiaStrain A. In some embodiments, all or substantially all of the bacteriain the bacterial formulation are Blautia Strain A. In some embodiments,the bacterial formulation comprises at least 1×10⁵, 5×10⁵, 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸ or 1×10⁹ colony forming units ofBlautia Strain A.

In some embodiments, the bacterial composition is administered orally,intravenously, intratumorally, or subcutaneously. In some embodiments,the bacterial composition is administered in 2 or more (e.g., 3 or more,4 or more or 5 or more doses). In some embodiments, the administrationto the subject of the two or more doses are separated by at least 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16hours, 17 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days.In some embodiments, a second bacterium is administered as part of anecological consortium.

In some embodiments, the method further comprises administering to thesubject an antibiotic. In some embodiments, the method further comprisesadministering to the subject one or more other cancer therapies. In someembodiments, the other cancer therapy is the surgical removal of atumor, the administration of a chemotherapeutic agent, theadministration of radiation therapy, the administration of anantibiotic, the administration of a cancer immunotherapy (e.g., animmune checkpoint inhibitor, a cancer-specific antibody, a cancervaccine, a primed antigen presenting cell, a cancer-specific T cell, acancer-specific chimeric antigen receptor (CAR) T cell, an immuneactivating protein, an adjuvant), and/or the administration of anothertherapeutic bacterium.

In some embodiments, the subject is a mammal. In some embodiments, thesubject is a human. In some embodiments, the subject is a non-humanmammal (e.g., a dog, a cat, a cow, a horse, a pig, a donkey, a goat, acamel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, agorilla or a chimpanzee).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that in a mouse colorectal carcinoma model, the efficacy oforally administered Blautia Strain A compared to that ofintraperitoneally (i.p.) administered anti-PD-1.

FIG. 2 shows inhibition of tumor growth (by volume) by the oraladministration of Blautia Strain A compared to intraperitoneally (i.p.)administered anti-PD-1 in a mouse colorectal carcinoma model.

FIG. 3 shows that in a mouse melanoma model, the efficacy of orallyadministered Blautia Strain A is compared to that of intraperitoneally(i.p.) administered anti-PD-L1.

FIG. 4 shows inhibition of tumor growth (by volume) by the oraladministration of Blautia Strain A compared to intraperitoneally (i.p.)administered anti-PD-L1 in a mouse melanoma model.

FIG. 5 shows that in a mouse melanoma model, the efficacy ofadministered combination therapy of Blautia massiliensis Strain A andanti-PD-L1.

FIG. 6 shows that in a mouse colorectal carcinoma model, theinfiltration of CD3+ immune cells was significantly increased in theanti-PD-1 group and the Blautia massiliensis Strain A group relative tothe vehicle group.

FIG. 7 shows that in a mouse melanoma model, the infiltration of CD3+immune cells was significantly increased in the anti-PD-L1 group and theBlautia massiliensis Strain A group relative to the vehicle group.

FIG. 8 shows that in a mouse colorectal carcinoma model, Blautiamassiliensis Strain A induced a striking upregulation of MHC Class Iexpression relative to the vehicle group.

FIG. 9 shows anaerobic bioreactor process at controlled pH at differentset points.

FIG. 10 shows total base addition during fermentation processes; samplesS1 and S3 were taken at designated points.

FIG. 11 shows cell recovery after downstream processing.

FIG. 12 shows relative cell viability of samples S1 and S3 afterdownstream processing.

DETAILED DESCRIPTION General

In certain aspects, provided herein are methods of treating cancer in asubject comprising administering to the subject a bacterial compositioncomprising Blautia Strain A.

Definitions

“Adjuvant” or “Adjuvant therapy” broadly refers to an agent that affectsan immunological or physiological response in a patient or subject. Forexample, an adjuvant might increase the presence of an antigen over timeor to an area of interest like a tumor, help absorb an antigenpresenting cell antigen, activate macrophages and lymphocytes andsupport the production of cytokines. By changing an immune response, anadjuvant might permit a smaller dose of an immune interacting agent toincrease the effectiveness or safety of a particular dose of the immuneinteracting agent. For example, an adjuvant might prevent T cellexhaustion and thus increase the effectiveness or safety of a particularimmune interacting agent.

“Administration” broadly refers to a route of administration of acomposition to a subject. Examples of routes of administration includeoral administration, rectal administration, topical administration,inhalation (nasal) or injection. Administration by injection includesintravenous (IV), intramuscular (IM), intratumoral (IT) and subcutaneous(SC) administration. The pharmaceutical compositions described hereincan be administered in any form by any effective route, including butnot limited to intratumoral, oral, parenteral, enteral, intravenous,intraperitoneal, topical, transdermal (e.g., using any standard patch),intradermal, ophthalmic, (intra)nasally, local, non-oral, such asaerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual,(trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal(e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal(e.g., trans- and perivaginally), intravesical, intrapulmonary,intraduodenal, intragastrical, and intrabronchial. In preferredembodiments, the pharmaceutical compositions described herein areadministered orally, rectally, intratumorally, topically,intravesically, by injection into or adjacent to a draining lymph node,intravenously, by inhalation or aerosol, or subcutaneously.

As used herein, the term “antibody” may refer to both an intact antibodyand an antigen binding fragment thereof. Intact antibodies areglycoproteins that include at least two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chain includesa heavy chain variable region (abbreviated herein as V_(H)) and a heavychain constant region. Each light chain includes a light chain variableregion (abbreviated herein as V_(L)) and a light chain constant region.The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The term “antibody” includes, for example, monoclonalantibodies, polyclonal antibodies, chimeric antibodies, humanizedantibodies, human antibodies, multispecific antibodies (e.g., bispecificantibodies), single-chain antibodies and antigen-binding antibodyfragments.

The terms “antigen binding fragment” and “antigen-binding portion” of anantibody, as used herein, refers to one or more fragments of an antibodythat retain the ability to bind to an antigen. Examples of bindingfragments encompassed within the term “antigen-binding fragment” of anantibody include Fab, Fab′, F(ab′)2, Fv, scFv, disulfide linked Fv, Fd,diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, andother antibody fragments that retain at least a portion of the variableregion of an intact antibody. These antibody fragments can be obtainedusing conventional recombinant and/or enzymatic techniques and can bescreened for antigen binding in the same manner as intact antibodies.

“Cancer” broadly refers to an uncontrolled, abnormal growth of a host'sown cells leading to invasion of surrounding tissue and potentiallytissue distal to the initial site of abnormal cell growth in the host.Major classes include carcinomas which are cancers of the epithelialtissue (e.g., skin, squamous cells); sarcomas which are cancers of theconnective tissue (e.g., bone, cartilage, fat, muscle, blood vessels,etc.); leukemias which are cancers of blood forming tissue (e.g., bonemarrow tissue); lymphomas and myelomas which are cancers of immunecells; and central nervous system cancers which include cancers frombrain and spinal tissue. “Cancer(s),” “neoplasm(s),” and “tumor(s)” areused herein interchangeably. As used herein, “cancer” refers to alltypes of cancer or neoplasm or malignant tumors including leukemias,carcinomas and sarcomas, whether new or recurring. Specific examples ofcancers are: carcinomas, sarcomas, myelomas, leukemias, lymphomas andmixed type tumors. Non-limiting examples of cancers are new or recurringcancers of the brain, melanoma, bladder, breast, cervix, colon, head andneck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate,sarcoma, stomach, uterus and medulloblastoma. Pediatric and adult tumorsinclude, but not limited to, those of bladder, brain, breast, bone,cervix, colon, connective tissue, fat, head and neck, kidney, liver,lung, mesothelium, melanocytes (melanoma), muscle, ovary, pancreas,prostate, stomach, small intestine, and uterus

The term “decrease” or “deplete” means a change, such that thedifference is, depending on circumstances, at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000or undetectable after treatment when compared to a pre-treatment state.

The term “ecological consortium” is a group of bacteria which tradesmetabolites and positively co-regulates one another, in contrast to twobacteria which induce host synergy through activating complementary hostpathways for improved efficacy.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chains.Certain epitopes can be defined by a particular sequence of amino acidsto which an antibody is capable of binding.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. The term “gene” applies to a specificgenomic sequence, as well as to a cDNA or an mRNA encoded by thatgenomic sequence.

“Identity” as between nucleic acid sequences of two nucleic acidmolecules can be determined as a percentage of identity using knowncomputer algorithms such as the “FASTA” program, using for example, thedefault parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci.USA 85:2444 (other programs include the GCG program package (Devereux,J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN,FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to HugeComputers, Mrtin J. Bishop, ed., Academic Press, San Diego, 1994, andCarillo et al. (1988) SIAM J Applied Math 48:1073). For example, theBLAST function of the National Center for Biotechnology Informationdatabase can be used to determine identity. Other commercially orpublicly available programs include, DNAStar “MegAlign” program(Madison, Wis.) and the University of Wisconsin Genetics Computer Group(UWG) “Gap” program (Madison Wis.)).

“Immunotherapy” is treatment that uses a subject's immune system totreat cancer and includes, for example, checkpoint inhibitors, cancervaccines, cytokines, cell therapy, CAR-T cells, and dendritic celltherapy.

The term “increase” means a change, such that the difference is,depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 2-fold, 4-fold, 10-fold, 100-fold, 10{circumflex over ( )}3fold, 10{circumflex over ( )}4 fold, 10{circumflex over ( )}5 fold,10{circumflex over ( )}6 fold, and/or 10{circumflex over ( )}7 foldgreater after treatment when compared to a pre-treatment state.Properties that may be increased include immune cells, bacterial cells,stromal cells, myeloid derived suppressor cells, fibroblasts,metabolites, and cytokines.

“Innate immune agonists” or “immuno-adjuvants” are small molecules,proteins, or other agents that specifically target innate immunereceptors including Toll-Like Receptors, NOD receptors, STING Pathwaycomponents. For example, LPS is a TLR-4 agonist that is bacteriallyderived or synthesized and aluminum can be used as an immune stimulatingadjuvant. immuno-adjuvants are a specific class of broader adjuvant oradjuvant therapy.

The term “isolated” or “enriched” encompasses a microbe, bacteria orother entity or substance that has been (1) separated from at least someof the components with which it was associated when initially produced(whether in nature or in an experimental setting), and/or (2) produced,prepared, purified, and/or manufactured by the hand of man. Isolatedmicrobes may be separated from at least about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, ormore of the other components with which they were initially associated.In some embodiments, isolated microbes are more than about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.As used herein, a substance is “pure” if it is substantially free ofother components. The terms “purify,” “purifying” and “purified” referto a microbe or other material that has been separated from at leastsome of the components with which it was associated either wheninitially produced or generated (e.g., whether in nature or in anexperimental setting), or during any time after its initial production.A microbe or a microbial population may be considered purified if it isisolated at or after production, such as from a material or environmentcontaining the microbe or microbial population, and a purified microbeor microbial population may contain other materials up to about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, or above about 90% and still be considered “isolated.”In some embodiments, purified microbes or microbial population are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. In the instance of microbial compositionsprovided herein, the one or more microbial types present in thecomposition can be independently purified from one or more othermicrobes produced and/or present in the material or environmentcontaining the microbial type. Microbial compositions and the microbialcomponents thereof are generally purified from residual habitatproducts.

As used herein, a gene is “overexpressed” in a bacteria if it isexpressed at a higher level in an engineered bacteria under at leastsome conditions than it is expressed by a wild-type bacteria of the samespecies under the same conditions. Similarly, a gene is “underexpressed”in a bacteria if it is expressed at a lower level in an engineeredbacteria under at least some conditions than it is expressed by awild-type bacteria of the same species under the same conditions.

The terms “polynucleotide” and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function. The following are non-limiting examples ofpolynucleotides: coding or non-coding regions of a gene or genefragment, loci (locus) defined from linkage analysis, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. A polynucleotide may be furthermodified, such as by conjugation with a labeling component. In allnucleic acid sequences provided herein, U nucleotides areinterchangeable with T nucleotides.

“Operational taxonomic units” and “OTU(s)” refer to a terminal leaf in aphylogenetic tree and is defined by a nucleic acid sequence, e.g., theentire genome, or a specific genetic sequence, and all sequences thatshare sequence identity to this nucleic acid sequence at the level ofspecies. In some embodiments the specific genetic sequence may be the16S sequence or a portion of the 16S sequence. In other embodiments, theentire genomes of two entities are sequenced and compared. In anotherembodiment, select regions such as multilocus sequence tags (MLST),specific genes, or sets of genes may be genetically compared. For 16S,OTUs that share ≥97% average nucleotide identity across the entire 16Sor some variable region of the 16S are considered the same OTU. See e.g.Claesson M J, Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R, Ross R P,and O'Toole P W. 2010. Comparison of two next-generation sequencingtechnologies for resolving highly complex microbiota composition usingtandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200.Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterialspecies definition in the genomic era. Philos Trans R Soc Lond B BiolSci 361: 1929-1940. For complete genomes, MLSTs, specific genes, otherthan 16S, or sets of genes OTUs that share ≥95% average nucleotideidentity are considered the same OTU. See e.g., Achtman M, and Wagner M.2008. Microbial diversity and the genetic nature of microbial species.Nat. Rev. Microbiol. 6: 431-440. Konstantinidis K T, Ramette A, andTiedje J M. 2006. The bacterial species definition in the genomic era.Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. OTUs are frequentlydefined by comparing sequences between organisms. Generally, sequenceswith less than 95% sequence identity are not considered to form part ofthe same OTU. OTUs may also be characterized by any combination ofnucleotide markers or genes, in particular highly conserved genes (e.g.,“house-keeping” genes), or a combination thereof. Operational TaxonomicUnits (OTUs) with taxonomic assignments made to, e.g., genus, species,and phylogenetic clade are provided herein.

As used herein, “specific binding” refers to the ability of an antibodyto bind to a predetermined antigen or the ability of a polypeptide tobind to its predetermined binding partner. Typically, an antibody orpolypeptide specifically binds to its predetermined antigen or bindingpartner with an affinity corresponding to a K_(D) of about 10⁻⁷ M orless, and binds to the predetermined antigen/binding partner with anaffinity (as expressed by K_(D)) that is at least 10 fold less, at least100 fold less or at least 1000 fold less than its affinity for bindingto a non-specific and unrelated antigen/binding partner (e.g., BSA,casein). Alternatively, specific binding applies more broadly to a twocomponent system where one component is a protein, lipid, orcarbohydrate or combination thereof and engages with the secondcomponent which is a protein, lipid, carbohydrate or combination thereofin a specific way.

The terms “subject” or “patient” refers to any animal. A subject or apatient described as “in need thereof” refers to one in need of atreatment for a disease. Mammals (i.e., mammalian animals) includehumans, laboratory animals (e.g., primates, rats, mice), livestock(e.g., cows, sheep, goats, pigs), and household pets (e.g., dogs, cats,rodents). For example, the subject may be a non-human mammal includingbut not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, agoat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey,a gorilla or a chimpanzee. The subject or patient may be healthy, or maybe suffering from a neoplasm at any developmental stage, wherein any ofthe stages are either caused by or opportunistically supported of acancer associated or causative pathogen, or may be at risk of developinga neoplasm, or transmitting to others a cancer associated or cancercausative pathogen. In some embodiments patients have lung cancer,bladder cancer, prostate cancer, ovarian cancer, and/or melanoma. Thepatients may have tumors that show enhanced macropinocytosis with theunderlying genomics of this process including Ras activation. In otherembodiments patients suffer from other cancers. In some embodiments, thesubject has undergone a cancer therapy.

“Strain” refers to a member of a bacterial species with a geneticsignature such that it may be differentiated from closely-relatedmembers of the same bacterial species. The genetic signature may be theabsence of all or part of at least one gene, the absence of all or partof at least on regulatory region (e.g., a promoter, a terminator, ariboswitch, a ribosome binding site), the absence (“curing”) of at leastone native plasmid, the presence of at least one recombinant gene, thepresence of at least one mutated gene, the presence of at least oneforeign gene (a gene derived from another species), the presence atleast one mutated regulatory region (e.g., a promoter, a terminator, ariboswitch, a ribosome binding site), the presence of at least onenon-native plasmid, the presence of at least one antibiotic resistancecassette, or a combination thereof. Genetic signatures between differentstrains may be identified by PCR amplification optionally followed byDNA sequencing of the genomic region(s) of interest or of the wholegenome. In the case in which one strain (compared with another of thesame species) has gained or lost antibiotic resistance or gained or losta biosynthetic capability (such as an auxotrophic strain), strains maybe differentiated by selection or counter-selection using an antibioticor nutrient/metabolite, respectively.

As used herein, the term “treating” a disease in a subject or “treating”a subject having or suspected of having a disease refers to subjectingthe subject to a pharmaceutical treatment, e.g., the administration ofone or more agents, such that at least one symptom of the disease isdecreased or prevented from worsening. Thus, in one embodiment,“treating” refers inter alia to delaying progression, expeditingremission, inducing remission, augmenting remission, speeding recovery,increasing efficacy of or decreasing resistance to alternativetherapeutics, or a combination thereof. In certain embodiments, a canceris treated if the subject experiences a reduction in tumor size, areduced number of tumors, a reduction in tumor growth, a reduction incancer metastasis and/or a reduced number of total cancer cellsfollowing treatment than would be expected in the absence of treatment.

Bacteria

In certain aspects, provided herein are methods of using a bacterialcomposition comprising Blautia Strain A. In some embodiments, theBlautia Strain A is Blautia massiliensis Strain A (ATCC Deposit NumberPTA-125134). In some embodiments, the Blautia Strain A is a straincomprising at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity (e.g., at least 99.5% sequence identity, atleast 99.6% sequence identity, at least 99.7% sequence identity, atleast 99.8% sequence identity, at least 99.9% sequence identity) to thenucleotide sequence of the Blautia Strain A.

Under the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purpose of Patent Procedure,the Blautia Strain A was deposited on Jun. 7, 2018, with the AmericanType Culture Collection (ATCC) of 10801 University Boulevard, Manassas,Va. 20110-2209 USA and was assigned ATCC Accession Number PTA-125134.

Applicant represents that the ATCC is a depository affording permanenceof the deposit and ready accessibility thereto by the public if a patentis granted. All restrictions on the availability to the public of thematerial so deposited will be irrevocably removed upon the granting of apatent. The material will be available during the pendency of the patentapplication to one determined by the Commissioner to be entitled theretounder 37 CFR 1.14 and 35 U.S.C. 122. The deposited material will bemaintained with all the care necessary to keep it viable anduncontaminated for a period of at least five years after the most recentrequest for the furnishing of a sample of the deposited plasmid, and inany case, for a period of at least thirty (30) years after the date ofdeposit or for the enforceable life of the patent, whichever period islonger. Applicant acknowledges its duty to replace the deposit shouldthe depository be unable to furnish a sample when requested due to thecondition of the deposit.

In some embodiments, the bacteria described herein are modified toimprove colonization and/or engraftment in the mammaliangastrointestinal tract (e.g., modified metabolism, such as improvedmucin degradation, enhanced competition profile, increased motility,increased adhesion to gut epithelial cells, modified chemotaxis). Insome embodiments, the bacteria described herein are modified to enhancetheir immunomodulatory and/or therapeutic effect (e.g., either alone orin combination with another therapeutic agent). In some embodiments, thebacteria described herein are modified to enhance immune activation(e.g., through modified production of polysaccharides, pili, fimbriae,adhesins, outer membrane vesicles). In some embodiments, the bacteriadescribed herein are modified to improve bacterial manufacturing (e.g.,higher oxygen tolerance, improved freeze-thaw tolerance, shortergeneration times).

Blautia Strain A can be cultured according to methods known in the art.For example, Blautia can be grown in ATCC Medium 2722, ATCC Medium 1490,or other medium using methods disclosed, for example in Caballero etal., 2017. “Cooperating Commensals Restore Colonization Resistance toVancomycin-Resistant Enterococcus faecium” Cell Host & Microbe21:592-602, which is hereby incorporated by reference in its entirety.

Bacterial Compositions

In certain aspects, provided herein are bacterial compositionscomprising Blautia Strain A. In some embodiments, the Blautia Strain Ais Blautia massiliensis Strain A (ATCC Deposit Number PTA-125134). Insome embodiments, the Blautia Strain A is a strain comprising at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity (e.g., at least 99.5% sequence identity, at least 99.6%sequence identity, at least 99.7% sequence identity, at least 99.8%sequence identity, at least 99.9% sequence identity) to the nucleotidesequence of the Blautia Strain A. In some embodiments, the bacterialformulation comprises a bacterium and/or a combination of bacteriadescribed herein and a pharmaceutically acceptable carrier.

In certain embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bacteria inthe bacterial composition are Blautia Strain A. In certain embodiments,substantially all of the bacteria in the bacterial composition areBlautia Strain A. In certain embodiments, the bacterial compositioncomprises at least 1×10³ colony forming units (CFUs), 1×10⁴ colonyforming units (CFUs), 1×10⁵ colony forming units (CFUs), 5×10⁵ colonyforming units (CFUs), 1×10⁶ colony forming units (CFUs), 2×10⁶ colonyforming units (CFUs), 3×10⁶ colony forming units (CFUs), 4×10⁶ colonyforming units (CFUs), 5×10⁶ colony forming units (CFUs), 6×10⁶ colonyforming units (CFUs), 7×10⁶ colony forming units (CFUs), 8×10⁶ colonyforming units (CFUs), 9×10⁶ colony forming units (CFUs), 1×10⁷ colonyforming units (CFUs), 2×10⁷ colony forming units (CFUs), 3×10⁷ colonyforming units (CFUs), 4×10⁷ colony forming units (CFUs), 5×10⁷ colonyforming units (CFUs), 6×10⁷ colony forming units (CFUs), 7×10⁷ colonyforming units (CFUs), 8×10⁷ colony forming units (CFUs), 9×10⁷ colonyforming units (CFUs), 1×10⁸ colony forming units (CFUs), 2×10⁸ colonyforming units (CFUs), 3×10⁸ colony forming units (CFUs), 4×10⁸ colonyforming units (CFUs), 5×10⁸ colony forming units (CFUs), 6×10⁸ colonyforming units (CFUs), 7×10⁸ colony forming units (CFUs), 8×10⁸ colonyforming units (CFUs), 9×10⁸ colony forming units (CFUs), 1×10⁹ colonyforming units (CFUs), 5×10⁹ colony forming units (CFUs), 1×10¹⁰ colonyforming units (CFUs) 5×10¹⁰ colony forming units (CFUs), 1×10¹¹ colonyforming units (CFUs) 5×10¹¹ colony forming units (CFUs), 1×10¹² colonyforming units (CFUs) 5×10¹² colony forming units (CFUs), 1×10¹³ colonyforming units (CFUs) of Blautia Strain A.

In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%of the bacteria in the composition are selected from among the bacterialspecies described herein. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of thebacteria in the composition are selected from among the bacterialstrains described herein.

In some embodiments, the compositions described herein may include onlyone species of bacteria described herein or may include two or morespecies of the bacteria described herein. For example, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the speciesdescribed herein, in any combination, can be included in thecompositions provided herein.

As described in detail below, the pharmaceutical compositions disclosedherein may be specially formulated for administration in solid or liquidform, including those adapted for oral or rectal administration.

In some embodiments, the composition described herein may be apharmaceutical composition, a dietary supplement, or a food product(e.g., a food or beverage). In some embodiments, the food product is ananimal feed.

In certain embodiments, the pharmaceutical composition for oraladministration described herein comprises an additional component thatenables efficient delivery of the bacteria to the colon. In someembodiments, pharmaceutical preparation that enables the delivery of thebacteria to the colon can be used. Examples of such formulations includepH sensitive compositions, such as buffered sachet formulations orenteric polymers that release their contents when the pH becomesalkaline after the enteric polymers pass through the stomach. When a pHsensitive composition is used for formulating the pharmaceuticalpreparation, the pH sensitive composition can be a polymer whose pHthreshold of the decomposition of the composition is between about 6.8and about 7.5.

Another embodiment of a pharmaceutical composition useful for deliveryof the bacteria to the colon is one that ensures the delivery to thecolon by delaying the release of the bacteria by approximately 3 to 5hours, which corresponds to the small intestinal transit time. In someembodiments, the pharmaceutical composition for delayed release includesa hydrogel shell. The hydrogel is hydrated and swells upon contact withgastrointestinal fluid, with the result that the contents areeffectively released (released predominantly in the colon). Delayedrelease dosage units include bacteria-containing compositions having amaterial which coats or selectively coats the bacteria. Examples of sucha selective coating material include in vivo degradable polymers,gradually hydrolyzable polymers, gradually water-soluble polymers,and/or enzyme degradable polymers. A wide variety of coating materialsfor efficiently delaying the release is available and includes, forexample, cellulose-based polymers such as hydroxypropyl cellulose,acrylic acid polymers and copolymers such as methacrylic acid polymersand copolymers, and vinyl polymers and copolymers such aspolyvinylpyrrolidone.

Examples of composition enabling the delivery to the colon furtherinclude bioadhesive compositions which specifically adhere to thecolonic mucosal membrane (for example, a polymer described in thespecification of U.S. Pat. No. 6,368,586, hereby incorporated byreference) and compositions into which a protease inhibitor isincorporated for protecting particularly a biopharmaceutical preparationin the gastrointestinal tracts from decomposition due to an activity ofa protease.

An example of a system enabling the delivery to the colon is a system ofdelivering a composition to the colon by pressure change in such a waythat the contents are released by utilizing pressure change caused bygeneration of gas in bacterial fermentation at a distal portion of thestomach. Such a system is not particularly limited, and a more specificexample thereof is a capsule which has contents dispersed in asuppository base and which is coated with a hydrophobic polymer (forexample, ethyl cellulose).

Another example of the system enabling the delivery to the colon is asystem of delivering a composition to the colon, the system beingspecifically decomposed by an enzyme (for example, a carbohydratehydrolase or a carbohydrate reductase) present in the colon. Such asystem is not particularly limited, and more specific examples thereofinclude systems which use food components such as non-starchpolysaccharides, amylose, xanthan gum, and azopolymers.

In some embodiments, formulations containing Blautia Strain A areprovided as encapsulated, enteric coated, or powder forms, with dosesranging up to 10¹¹ cfu (e.g., up to 10¹⁰ cfu). In some embodiments, thecomposition comprises 5×10¹¹ cfu of Blautia Strain A and 10% (w/w) cornstarch in a capsule. The capsule is enteric coated for duodenal releaseat pH5.5 In some embodiments, the capsule is enteric coated for duodenalrelease at pH 5.5. In some embodiments, the composition comprises apowder of freeze-dried Blautia Strain A which is deemed “QualifiedPresumption of Safety” (QPS) status. In some embodiments, thecomposition is stable at frozen or refrigerated temperature.

Methods for producing microbial compositions may include three mainprocessing steps. The steps are: organism banking, organism production,and preservation. In certain embodiments, a sample that contains anabundance of Blautia Strain A may be cultured by avoiding an isolationstep.

For banking, the strains included in the microbial composition may be(1) isolated directly from a specimen or taken from a banked stock, (2)optionally cultured on a nutrient agar or broth that supports growth togenerate viable biomass, and (3) the biomass optionally preserved inmultiple aliquots in long-term storage.

In embodiments using a culturing step, the agar or broth may containnutrients that provide essential elements and specific factors thatenable growth. An example would be a medium composed of 20 g/L glucose,10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/Lsodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/Lmagnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1mg/L menadione. Another example would be a medium composed of 10 g/Lbeef extract, 10 g/L peptone, 5 g/L sodium chloride, 5 g/L dextrose, 3g/L yeast extract, 3 g/L sodium acetate, 1 g/L soluble starch, and 0.5g/L L-cysteine HCl, at pH 6.8. A variety of microbiological media andvariations are well known in the art (e.g., R. M. Atlas, Handbook ofMicrobiological Media (2010) CRC Press). Culture media can be added tothe culture at the start, may be added during the culture, or may beintermittently/continuously flowed through the culture. The strains inthe bacterial composition may be cultivated alone, as a subset of themicrobial composition, or as an entire collection comprising themicrobial composition. As an example, a first strain may be cultivatedtogether with a second strain in a mixed continuous culture, at adilution rate lower than the maximum growth rate of either cell toprevent the culture from washing out of the cultivation.

The inoculated culture is incubated under favorable conditions for atime sufficient to build biomass. For microbial compositions for humanuse this is often at 37° C. temperature, pH, and other parameter withvalues similar to the normal human niche. The environment may beactively controlled, passively controlled (e.g., via buffers), orallowed to drift. For example, for anaerobic bacterial compositions, ananoxic/reducing environment may be employed. This can be accomplished byaddition of reducing agents such as cysteine to the broth, and/orstripping it of oxygen. As an example, a culture of a bacterialcomposition may be grown at 37° C., pH 7, in the medium above,pre-reduced with 1 g/L cysteine-HCl.

When the culture has generated sufficient biomass, it may be preservedfor banking. The organisms may be placed into a chemical milieu thatprotects from freezing (adding ‘cryoprotectants’), drying(‘lyoprotectants’), and/or osmotic shock (‘osmoprotectants’), dispensinginto multiple (optionally identical) containers to create a uniformbank, and then treating the culture for preservation. Containers aregenerally impermeable and have closures that assure isolation from theenvironment. Cryopreservation treatment is accomplished by freezing aliquid at ultra-low temperatures (e.g., at or below −80° C.). Driedpreservation removes water from the culture by evaporation (in the caseof spray drying or ‘cool drying’) or by sublimation (e.g., for freezedrying, spray freeze drying). Removal of water improves long-termmicrobial composition storage stability at temperatures elevated abovecryogenic conditions. If the microbial composition comprises, forexample, spore forming species and results in the production of spores,the final composition may be purified by additional means such asdensity gradient centrifugation and preserved using the techniques[?]described above[?]. Microbial composition banking may be done byculturing and preserving the strains individually, or by mixing thestrains together to create a combined bank. As an example ofcryopreservation, a microbial composition culture may be harvested bycentrifugation to pellet the cells from the culture medium, thesupernatant decanted and replaced with fresh culture broth containing15% glycerol. The culture can then be aliquoted into 1 mL cryotubes,sealed, and placed at −80° C. for long-term viability retention. Thisprocedure achieves acceptable viability upon recovery from frozenstorage.

Microbial production may be conducted using similar culture steps tobanking, including medium composition and culture conditions describedabove. It may be conducted at larger scales of operation, especially forclinical development or commercial production. At larger scales, theremay be several subcultivations of the microbial composition prior to thefinal cultivation. At the end of cultivation, the culture is harvestedto enable further formulation into a dosage form for administration.This can involve concentration, removal of undesirable mediumcomponents, and/or introduction into a chemical milieu that preservesthe microbial composition and renders it acceptable for administrationvia the chosen route. For example, a microbial composition may becultivated to a concentration of 10¹⁰ CFU/mL, then concentrated 20-foldby tangential flow microfiltration; the spent medium may be exchanged bydiafiltering with a preservative medium consisting of 2% gelatin, 100 mMtrehalose, and 10 mM sodium phosphate buffer. The suspension can then befreeze-dried to a powder and titrated.

After drying, the powder may be blended to an appropriate potency, andmixed with other cultures and/or a filler such as microcrystallinecellulose for consistency and ease of handling, and the bacterialcomposition formulated as provided herein.

In certain aspects, provided are bacterial compositions foradministration subjects. In some embodiments, the bacterial compositionsare combined with additional active and/or inactive materials in orderto produce a final product, which may be in single dosage unit or in amulti-dose format.

In some embodiments, the composition comprises at least onecarbohydrate. A “carbohydrate” refers to a sugar or polymer of sugars.The terms “saccharide,” “polysaccharide,” “carbohydrate,” and“oligosaccharide” may be used interchangeably. Most carbohydrates arealdehydes or ketones with many hydroxyl groups, usually one on eachcarbon atom of the molecule. Carbohydrates generally have the molecularformula C_(n)H_(2n)O_(n). A carbohydrate may be a monosaccharide, adisaccharide, trisaccharide, oligosaccharide, or polysaccharide. Themost basic carbohydrate is a monosaccharide, such as glucose, sucrose,galactose, mannose, ribose, arabinose, xylose, and fructose.Disaccharides are two joined monosaccharides. Exemplary disaccharidesinclude sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and six monosaccharide units(e.g., raffinose, stachyose), and polysaccharides include six or moremonosaccharide units. Exemplary polysaccharides include starch,glycogen, and cellulose. Carbohydrates may contain modified saccharideunits such as 2′-deoxyribose wherein a hydroxyl group is removed,2′-fluororibose wherein a hydroxyl group is replaced with a fluorine, orN-acetylglucosamine, a nitrogen-containing form of glucose (e.g.,2′-fluororibose, deoxyribose, and hexose). Carbohydrates may exist inmany different forms, for example, conformers, cyclic forms, acyclicforms, stereoisomers, tautomers, anomers, and isomers.

In some embodiments, the composition comprises at least one lipid. Asused herein, a “lipid” includes fats, oils, triglycerides, cholesterol,phospholipids, fatty acids in any form including free fatty acids. Fats,oils and fatty acids can be saturated, unsaturated (cis or trans) orpartially unsaturated (cis or trans). In some embodiments the lipidcomprises at least one fatty acid selected from lauric acid (12:0),myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1),margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0),oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3),octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid(20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4),eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoicacid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6)(DHA), and tetracosanoic acid (24:0). In some embodiments thecomposition comprises at least one modified lipid, for example a lipidthat has been modified by cooking.

In some embodiments, the composition comprises at least one supplementalmineral or mineral source. Examples of minerals include, withoutlimitation: chloride, sodium, calcium, iron, chromium, copper, iodine,zinc, magnesium, manganese, molybdenum, phosphorus, potassium, andselenium. Suitable forms of any of the foregoing minerals includesoluble mineral salts, slightly soluble mineral salts, insoluble mineralsalts, chelated minerals, mineral complexes, non-reactive minerals suchas carbonyl minerals, and reduced minerals, and combinations thereof.

In some embodiments, the composition comprises at least one supplementalvitamin. The at least one vitamin can be fat-soluble or water solublevitamins. Suitable vitamins include but are not limited to vitamin C,vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin,vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenicacid, and biotin. Suitable forms of any of the foregoing are salts ofthe vitamin, derivatives of the vitamin, compounds having the same orsimilar activity of the vitamin, and metabolites of the vitamin.

In some embodiments, the composition comprises an excipient.Non-limiting examples of suitable excipients include a buffering agent,a preservative, a stabilizer, a binder, a compaction agent, a lubricant,a dispersion enhancer, a disintegration agent, a flavoring agent, asweetener, and a coloring agent.

In some embodiments, the excipient is a buffering agent. Non-limitingexamples of suitable buffering agents include sodium citrate, magnesiumcarbonate, magnesium bicarbonate, calcium carbonate, and calciumbicarbonate.

In some embodiments, the excipient comprises a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as alpha-tocopherol and ascorbate, and antimicrobials, such asparabens, chlorobutanol, and phenol.

In some embodiments, the composition comprises a binder as an excipient.Non-limiting examples of suitable binders include starches,pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose,methylcellulose, sodium carboxymethylcellulose, ethylcellulose,polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fattyacid alcohol, polyethylene glycol, polyols, saccharides,oligosaccharides, and combinations thereof.

In some embodiments, the composition comprises a lubricant as anexcipient. Non-limiting examples of suitable lubricants includemagnesium stearate, calcium stearate, zinc stearate, hydrogenatedvegetable oils, sterotex, polyoxyethylene monostearate, talc,polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesiumlauryl sulfate, and light mineral oil.

In some embodiments, the composition comprises a dispersion enhancer asan excipient. Non-limiting examples of suitable dispersants includestarch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin,bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose as high HLBemulsifier surfactants.

In some embodiments, the composition comprises a disintegrant as anexcipient. In some embodiments the disintegrant is a non-effervescentdisintegrant. Non-limiting examples of suitable non-effervescentdisintegrants include starches such as corn starch, potato starch,pregelatinized and modified starches thereof, sweeteners, clays, such asbentonite, microcrystalline cellulose, alginates, sodium starchglycolate, gums such as agar, guar, locust bean, karaya, pectin, andtragacanth. In some embodiments the disintegrant is an effervescentdisintegrant. Non-limiting examples of suitable effervescentdisintegrants include sodium bicarbonate in combination with citricacid, and sodium bicarbonate in combination with tartaric acid.

In some embodiments, the bacterial formulation comprises an entericcoating or micro encapsulation. In certain embodiments, the entericcoating or micro encapsulation improves targeting to a desired region ofthe gastrointestinal tract. For example, in certain embodiments, thebacterial composition comprises an enteric coating and/or microcapsulesthat dissolves at a pH associated with a particular region of thegastrointestinal tract. In some embodiments, the enteric coating and/ormicrocapsules dissolve at a pH of about 5.5-6.2 to release in theduodenum, at a pH value of about 7.2-7.5 to release in the ileum, and/orat a pH value of about 5.6-6.2 to release in the colon. Exemplaryenteric coatings and microcapsules are described, for example, in U.S.Pat. Pub. No. 2016/0022592, which is hereby incorporated by reference inits entirety.

In some embodiments, the composition is a food product (e.g., a food orbeverage) such as a health food or beverage, a food or beverage forinfants, a food or beverage for pregnant women, athletes, seniorcitizens or other specified group, a functional food, a beverage, a foodor beverage for specified health use, a dietary supplement, a food orbeverage for patients, or an animal feed. Specific examples of the foodsand beverages include various beverages such as juices, refreshingbeverages, tea beverages, drink preparations, jelly beverages, andfunctional beverages; alcoholic beverages such as beers;carbohydrate-containing foods such as rice food products, noodles,breads, and pastas; paste products such as fish hams, sausages, pasteproducts of seafood; retort pouch products such as curries, food dressedwith a thick starchy sauces, and Chinese soups; soups; dairy productssuch as milk, dairy beverages, ice creams, cheeses, and yogurts;fermented products such as fermented soybean pastes, yogurts, fermentedbeverages, and pickles; bean products; various confectionery products,including biscuits, cookies, and the like, candies, chewing gums,gummies, cold desserts including jellies, cream caramels, and frozendesserts; instant foods such as instant soups and instant soy-beansoups; microwavable foods; and the like. Further, the examples alsoinclude health foods and beverages prepared in the forms of powders,granules, tablets, capsules, liquids, pastes, and jellies.

In certain embodiments, the bacteria disclosed herein are administeredin conjunction with a prebiotic to the subject. Prebiotics arecarbohydrates which are generally indigestible by a host animal and areselectively fermented or metabolized by bacteria. Prebiotics may beshort-chain carbohydrates (e.g., oligosaccharides) and/or simple sugars(e.g., mono- and di-saccharides) and/or mucins (heavily glycosylatedproteins) that alter the composition or metabolism of a microbiome inthe host. The short chain carbohydrates are also referred to asoligosaccharides, and usually contain from 2 or 3 and up to 8, 9, 10, 15or more sugar moieties. When prebiotics are introduced to a host, theprebiotics affect the bacteria within the host and do not directlyaffect the host. In certain aspects, a prebiotic composition canselectively stimulate the growth and/or activity of one of a limitednumber of bacteria in a host. Prebiotics include oligosaccharides suchas fructooligosaccharides (FOS) (including inulin),galactooligosaccharides (GOS), trans-galactooligosaccharides,xylooligosaccharides (XOS), chitooligosaccharides (COS), soyoligosaccharides (e.g., stachyose and raffinose) gentiooligosaccharides,isomaltooligosaccharides, mannooligosaccharides, maltooligosaccharidesand mannanoligosaccharides. Oligosaccharides are not necessarily singlecomponents, and can be mixtures containing oligosaccharides withdifferent degrees of oligomerization, sometimes including the parentdisaccharide and the monomeric sugars. Various types of oligosaccharidesare found as natural components in many common foods, including fruits,vegetables, milk, and honey. Specific examples of oligosaccharides arelactulose, lactosucrose, palatinose, glycosyl sucrose, guar gum, gumArabic, tagalose, amylose, amylopectin, pectin, xylan, andcyclodextrins. Prebiotics may also be purified or chemically orenzymatically synthesized.

Administration

In certain aspects, provided herein is a method of delivering abacterium and/or a bacterial composition described herein to a subject.In some embodiments of the methods provided herein, the bacteria areadministered in conjunction with the administration of a cancertherapeutic. In some embodiments, the bacteria is co-formulated in apharmaceutical composition with the cancer therapeutic. In someembodiments, the bacteria is co-administered with the cancertherapeutic. In some embodiments, the cancer therapeutic is administeredto the subject before administration of the bacteria (e.g., about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutesbefore, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 days before). In some embodiments, the cancertherapeutic is administered to the subject after administration of thebacteria (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50 or 55 minutes after, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours after, or about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after). In someembodiments, the same mode of delivery is used to deliver both thebacteria and the cancer therapeutic. In some embodiments different modesof delivery are used to administer the bacteria and the cancertherapeutic. For example, in some embodiments, the bacteria isadministered orally while the cancer therapeutic is administered viainjection (e.g., an intravenous, intramuscular and/or intratumoralinjection).

In certain embodiments, the pharmaceutical compositions, dosage forms,and kits described herein can be administered in conjunction with anyother conventional anti-cancer treatment, such as, for example,radiation therapy and surgical resection of the tumor. These treatmentsmay be applied as necessary and/or as indicated and may occur before,concurrent with or after administration of the pharmaceuticalcompositions, dosage forms, and kits described herein.

The dosage regimen can be any of a variety of methods and amounts, andcan be determined by one skilled in the art according to known clinicalfactors. As is known in the medical arts, dosages for any one patientcan depend on many factors, including the subject's species, size, bodysurface area, age, sex, immunocompetence, and general health, theparticular microorganism to be administered, duration and route ofadministration, the kind and stage of the disease, for example, tumorsize, and other compounds such as drugs being administered concurrently.In addition to the above factors, such levels can be affected by theinfectivity of the microorganism, and the nature of the microorganism,as can be determined by one skilled in the art. In the present methods,appropriate minimum dosage levels of microorganisms can be levelssufficient for the microorganism to survive, grow and replicate in atumor or metastasis. The methods of treatment described herein may besuitable for the treatment of a primary tumor, a secondary tumor ormetastasis, as well as for recurring tumors or cancers. The dose of thepharmaceutical compositions described herein may be appropriately set oradjusted in accordance with the dosage form, the route ofadministration, the degree or stage of a target disease, and the like.For example, the general effective dose of the agents may range between0.01 mg/kg body weight/day and 1000 mg/kg body weight/day, between 0.1mg/kg body weight/day and 1000 mg/kg body weight/day, 0.5 mg/kg bodyweight/day and 500 mg/kg body weight/day, 1 mg/kg body weight/day and100 mg/kg body weight/day, or between 5 mg/kg body weight/day and 50mg/kg body weight/day. The effective dose may be 0.01, 0.05, 0.1, 0.5,1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000mg/kg body weight/day or more, but the dose is not limited thereto.

In some embodiments, the dose administered to a subject is sufficient toprevent cancer, delay its onset, or slow or stop its progression orprevent a relapse of a cancer. One skilled in the art will recognizethat dosage will depend upon a variety of factors including the strengthof the particular compound employed, as well as the age, species,condition, and body weight of the subject. The size of the dose willalso be determined by the route, timing, and frequency of administrationas well as the existence, nature, and extent of any adverse side-effectsthat might accompany the administration of a particular compound and thedesired physiological effect.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages, which are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. An effective dosage and treatment protocol canbe determined by routine and conventional means, starting e.g., with alow dose in laboratory animals and then increasing the dosage whilemonitoring the effects, and systematically varying the dosage regimen aswell. Animal studies are commonly used to determine the maximaltolerable dose (“MTD”) of bioactive agent per kilogram weight. Thoseskilled in the art regularly extrapolate doses for efficacy, whileavoiding toxicity, in other species, including humans.

In accordance with the above, in therapeutic applications, the dosagesof the active agents used in accordance with the invention varydepending on the active agent, the age, weight, and clinical conditionof the recipient patient, and the experience and judgment of theclinician or practitioner administering the therapy, among other factorsaffecting the selected dosage. Generally, the dose should be sufficientto result in slowing, and preferably regressing, the growth of thetumors and most preferably causing complete regression of the cancer.

Separate administrations can include any number of two or moreadministrations (e.g., doses), including two, three, four, five or sixadministrations. One skilled in the art can readily determine the numberof administrations to perform, or the desirability of performing one ormore additional administrations, according to methods known in the artfor monitoring therapeutic methods and other monitoring methods providedherein. In some embodiments, the doses may be separated by at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 days or 1, 2, 3, or 4 weeks.Accordingly, the methods provided herein include methods of providing tothe subject one or more administrations of a bacterium, where the numberof administrations can be determined by monitoring the subject, and,based on the results of the monitoring, determining whether or not toprovide one or more additional administrations. Deciding on whether ornot to provide one or more additional administrations can be based on avariety of monitoring results, including, but not limited to, indicationof tumor growth or inhibition of tumor growth, appearance of newmetastases or inhibition of metastasis, the subject's anti-bacteriumantibody titer, the subject's anti-tumor antibody titer, the overallhealth of the subject and/or the weight of the subject.

The time period between administrations can be any of a variety of timeperiods. The time period between administrations can be a function ofany of a variety of factors, including monitoring steps, as described inrelation to the number of administrations, the time period for a subjectto mount an immune response and/or the time period for a subject toclear the bacteria from normal tissue. In one example, the time periodcan be a function of the time period for a subject to mount an immuneresponse; for example, the time period can be more than the time periodfor a subject to mount an immune response, such as more than about oneweek, more than about ten days, more than about two weeks, or more thanabout a month; in another example, the time period can be less than thetime period for a subject to mount an immune response, such as less thanabout one week, less than about ten days, less than about two weeks, orless than about a month. In another example, the time period can be afunction of the time period for a subject to clear the bacteria fromnormal tissue; for example, the time period can be more than the timeperiod for a subject to clear the bacteria from normal tissue, such asmore than about a day, more than about two days, more than about threedays, more than about five days, or more than about a week.

In some embodiments, the delivery of a cancer therapeutic in combinationwith the bacteria described herein reduces the adverse effects and/orimproves the efficacy of the cancer therapeutic.

The effective dose of a cancer therapeutic described herein is theamount of the therapeutic agent that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, with the least toxicity to the patient. The effectivedosage level can be identified using the methods described herein andwill depend upon a variety of pharmacokinetic factors including theactivity of the particular compositions administered, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. In general, aneffective dose of a cancer therapy will be the amount of the therapeuticagent which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

The toxicity of a cancer therapy is the level of adverse effectsexperienced by the subject during and following treatment. Adverseevents associated with cancer therapy toxicity include, but are notlimited to, abdominal pain, acid indigestion, acid reflux, allergicreactions, alopecia, anaphylaxis, anemia, anxiety, lack of appetite,arthralgias, asthenia, ataxia, azotemia, loss of balance, bone pain,bleeding, blood clots, low blood pressure, elevated blood pressure,difficulty breathing, bronchitis, bruising, low white blood cell count,low red blood cell count, low platelet count, cardiotoxicity, cystitis,hemorrhagic cystitis, arrhythmias, heart valve disease, cardiomyopathy,coronary artery disease, cataracts, central neurotoxicity, cognitiveimpairment, confusion, conjunctivitis, constipation, coughing, cramping,cystitis, deep vein thrombosis, dehydration, depression, diarrhea,dizziness, dry mouth, dry skin, dyspepsia, dyspnea, edema, electrolyteimbalance, esophagitis, fatigue, loss of fertility, fever, flatulence,flushing, gastric reflux, gastroesophageal reflux disease, genital pain,granulocytopenia, gynecomastia, glaucoma, hair loss, hand-foot syndrome,headache, hearing loss, heart failure, heart palpitations, heartburn,hematoma, hemorrhagic cystitis, hepatotoxicity, hyperamylasemia,hypercalcemia, hyperchloremia, hyperglycemia, hyperkalemia,hyperlipasemia, hypermagnesemia, hypernatremia, hyperphosphatemia,hyperpigmentation, hypertriglyceridemia, hyperuricemia, hypoalbuminemia,hypocalcemia, hypochloremia, hypoglycemia, hypokalemia, hypomagnesemia,hyponatremia, hypophosphatemia, impotence, infection, injection sitereactions, insomnia, iron deficiency, itching, joint pain, kidneyfailure, leukopenia, liver dysfunction, memory loss, menopause, mouthsores, mucositis, muscle pain, myalgias, myelosuppression, myocarditis,neutropenic fever, nausea, nephrotoxicity, neutropenia, nosebleeds,numbness, ototoxicity, pain, palmar-plantar erythrodysesthesia,pancytopenia, pericarditis, peripheral neuropathy, pharyngitis,photophobia, photosensitivity, pneumonia, pneumonitis, proteinuria,pulmonary embolus, pulmonary fibrosis, pulmonary toxicity, rash, rapidheart beat, rectal bleeding, restlessness, rhinitis, seizures, shortnessof breath, sinusitis, thrombocytopenia, tinnitus, urinary tractinfection, vaginal bleeding, vaginal dryness, vertigo, water retention,weakness, weight loss, weight gain, and xerostomia. In general, toxicityis acceptable if the benefits to the subject achieved through thetherapy outweigh the adverse events experienced by the subject due tothe therapy.

In some embodiments, the administration of the bacterial compositiontreats the cancer. In some embodiments, the bacterial compositioninduces an anti-tumor immune response in the subject.

Therapeutic Agents

In certain aspects, the methods provided herein include theadministration to a subject of a bacterium and/or a bacterialcomposition described herein (e.g., a Blautia Strain A-containingbacterial composition) either alone or in combination with anothercancer therapeutic. The other cancer therapeutic may include e.g.,surgical resection, radiotherapy, or a cancer therapeutic agent. In someembodiments, the bacterial composition and the other cancer therapy canbe administered to the subject in any order. In some embodiments, thebacterial composition and the other cancer therapy are administeredconjointly.

In some embodiments the bacterium is administered to the subject beforethe cancer therapeutic is administered (e.g., at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 daysbefore). In some embodiments the bacterium is administered to thesubject after the cancer therapeutic is administered (e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23 or 24 hours after or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 days after). In some embodiments, the bacterium and the cancertherapeutic are administered to the subject simultaneously or nearlysimultaneously (e.g., administrations occur within an hour of eachother). In some embodiments, the subject is administered an antibioticbefore the bacterium is administered to the subject (e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 days before). In some embodiments, the subject is administered anantibiotic after the bacterium is administered to the subject (e.g., atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 days after). In some embodiments, the bacterium and theantibiotic are administered to the subject simultaneously or nearlysimultaneously (e.g., administrations occur within an hour of eachother).

In certain embodiments, the subject may undergo surgery. Types ofsurgery include but are not limited to preventative, diagnostic orstaging, curative and palliative surgery. Curative surgery is a cancertreatment that may be used in conjunction with other therapies, such asthe treatment of the present invention, chemotherapy, radiotherapy,hormonal therapy, gene therapy, immunotherapy and/or alternativetherapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and microscopically controlled surgery(Mohs' surgery). Upon excision of part of all of cancerous cells,tissue, or tumor, a cavity may be formed in the body.

In certain embodiments, the subject may undergo radiation therapy.Radiation therapy includes the administration or application of aradiotherapeutic agents and factors including but not limited to Inaddition to trays, UV-irradiation, microwaves, electronic emissions, andradioisotopes. The localized tumor site may be irradiated, including byone or more the above described forms of radiations. All of thesefactors may effect a broad range of damage DNA, on the precursors ofDNA, the replication and repair of DNA, and the assembly and maintenanceof chromosomes.

Dosage ranges for X-rays range from daily doses of 50 to 200 roentgensfor prolonged periods of time (3 to 4 weeks), to single doses of 2000 to6000 roentgens. Dosage ranges for radioisotopes vary widely, and dependon the half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells.

In certain aspects, the methods provided herein further compriseadministering another cancer therapeutic to the subject.

In some embodiments, the cancer therapeutic is a chemotherapeutic agent.Examples of such chemotherapeutic agents include, but are not limitedto, alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gammalI and calicheamicin omegal1; dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores, aclacinomysins,actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonicacid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes(especially T-2 toxin, verracurin A, roridin A and anguidine); urethan;vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide;thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumcoordination complexes such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11);topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

In some embodiments, the cancer therapeutic is a cancer immunotherapyagent. Immunotherapy refers to a treatment that uses a subject's immunesystem to treat cancer, e.g., checkpoint inhibitors, cancer vaccines,cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.Non-limiting examples of immunotherapies are checkpoint inhibitorsinclude Nivolumab (BMS, anti-PD-1), Pembrolizumab (Merck, anti-PD-1),Ipilimumab (BMS, anti-CTLA-4), MEDI4736 (AstraZeneca, anti-PD-L1), andMPDL3280A (Roche, anti-PD-L1). Other immunotherapies may be tumorvaccines, such as Gardail, Cervarix, BCG, sipulencel-T, Gp100:209-217,AGS-003, DCVax-L, Algenpantucel-L, Tergenpantucel-L, TG4010, ProstAtak,Prostvac-V/R-TRICOM, Rindopepimul, E75 peptide acetate, IMA901,POL-103A, Belagenpumatucel-L, GSK1572932A, MDX-1279, GV1001, andTecemotide. Immunotherapy may be administered via injection (e.g.,intravenously, intratumorally, subcutaneously, or into lymph nodes), butmay also be administered orally, topically, or via aerosol.Immunotherapies may comprise adjuvants such as cytokines.

In some embodiments, the immunotherapy agent is an immune checkpointinhibitor. Immune checkpoint inhibition broadly refers to inhibiting thecheckpoints that cancer cells can produce to prevent or downregulate animmune response. Examples of immune checkpoint proteins include, but arenot limited to, CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA,KIR, LAG3, TIM-3 or VISTA. Immune checkpoint inhibitors can beantibodies or antigen binding fragments thereof that bind to and inhibitan immune checkpoint protein. Examples of immune checkpoint inhibitorsinclude, but are not limited to, nivolumab, pembrolizumab, pidilizumab,AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736,MSB-0020718C, AUR-012 and STI-A1010.

In certain embodiments, immune checkpoint inhibitors can be aninhibitory nucleic acid molecule (e.g., an siRNA molecule, an shRNAmolecule or an antisense RNA molecule) that inhibits expression of animmune checkpoint protein that inhibits expression of an immunecheckpoint protein.

In some embodiments, the immune checkpoint inhibitor is a siRNAmolecule. Such siRNA molecules should include a region of sufficienthomology to the target region, and be of sufficient length in terms ofnucleotides, such that the siRNA molecule down-regulate target RNA(e.g., RNA of an immune checkpoint protein). The term “ribonucleotide”or “nucleotide” can, in the case of a modified RNA or nucleotidesurrogate, also refer to a modified nucleotide, or surrogate replacementmoiety at one or more positions. It is not necessary that there beperfect complementarity between the siRNA molecule and the target, butthe correspondence must be sufficient to enable the siRNA molecule todirect sequence-specific silencing, such as by RNAi cleavage of thetarget RNA. In some embodiments, the sense strand need only besufficiently complementary with the antisense strand to maintain theoverall double-strand character of the molecule.

In addition, an siRNA molecule may be modified or include nucleosidesurrogates. Single stranded regions of an siRNA molecule may be modifiedor include nucleoside surrogates, e.g., the unpaired region or regionsof a hairpin structure, e.g., a region which links two complementaryregions, can have modifications or nucleoside surrogates. Modificationto stabilize one or more 3′- or 5′-terminus of an siRNA molecule, e.g.,against exonucleases, or to favor the antisense siRNA agent to enterinto RISC are also useful. Modifications can include C3 (or C6, C7, C12)amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers(C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol),special biotin or fluorescein reagents that come as phosphoramidites andthat have another DMT-protected hydroxyl group, allowing multiplecouplings during RNA synthesis.

Each strand of an siRNA molecule can be equal to or less than 35, 30,25, 24, 23, 22, 21, or 20 nucleotides in length. In some embodiments,the strand is at least 19 nucleotides in length. For example, eachstrand can be between 21 and 25 nucleotides in length. In someembodiments, siRNA agents have a duplex region of 17, 18, 19, 29, 21,22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, such asone or two 3′ overhangs, of 2-3 nucleotides.

In some embodiments, the immune checkpoint inhibitor is a shRNAmolecule. A “small hairpin RNA” or “short hairpin RNA” or “shRNA”includes a short RNA sequence that makes a tight hairpin turn that canbe used to silence gene expression via RNA interference. The shRNAsprovided herein may be chemically synthesized or transcribed from atranscriptional cassette in a DNA plasmid. The shRNA hairpin structureis cleaved by the cellular machinery into siRNA, which is then bound tothe RNA-induced silencing complex (RISC).

In some embodiments, shRNAs are about 15-60, 15-50, or 15-40 (duplex)nucleotides in length, about 15-30, 15-25, or 19-25 (duplex) nucleotidesin length, or are about 20-24, 21-22, or 21-23 (duplex) nucleotides inlength (e.g., each complementary sequence of the double-stranded shRNAis 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, orabout 20-24, 21-22, or 21-23 nucleotides in length, and thedouble-stranded shRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or19-25 base pairs in length, or about 18-22, 19-20, or 19-21 base pairsin length). shRNA duplexes may comprise 3′ overhangs of about 1 to about4 nucleotides or about 2 to about 3 nucleotides on the antisense strandand/or 5′-phosphate termini on the sense strand. In some embodiments,the shRNA comprises a sense strand and/or antisense strand sequence offrom about 15 to about 60 nucleotides in length (e.g., about 15-60,15-55, 15-50, 15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides inlength), or from about 19 to about 40 nucleotides in length (e.g., about19-40, 19-35, 19-30, or 19-25 nucleotides in length), or from about 19to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23nucleotides in length).

Non-limiting examples of shRNA include a double-stranded polynucleotidemolecule assembled from a single-stranded molecule, where the sense andantisense regions are linked by a nucleic acid-based or non-nucleicacid-based linker; and a double-stranded polynucleotide molecule with ahairpin secondary structure having self-complementary sense andantisense regions. In some embodiments, the sense and antisense strandsof the shRNA are linked by a loop structure comprising from about 1 toabout 25 nucleotides, from about 2 to about 20 nucleotides, from about 4to about 15 nucleotides, from about 5 to about 12 nucleotides, or 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or more nucleotides.

Additional embodiments related to the shRNAs, as well as methods ofdesigning and synthesizing such shRNAs, are described in U.S. patentapplication publication number 2011/0071208, the disclosure of which isherein incorporated by reference in its entirety for all purposes.

In some embodiments, the immune checkpoint inhibitor is an antisenseoligonucleotide compounds that inhibits expression of an immunecheckpoint protein. In certain embodiments, the degree ofcomplementarity between the target sequence and antisense targetingsequence is sufficient to form a stable duplex. The region ofcomplementarity of the antisense oligonucleotides with the target RNAsequence may be as short as 8-11 bases, but can be 12-15 bases or more,e.g., 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases,or 15-20 bases, including all integers in between these ranges. Anantisense oligonucleotide of about 14-15 bases is generally long enoughto have a unique complementary sequence.

In certain embodiments, antisense oligonucleotides may be 100%complementary to the target sequence, or may include mismatches, e.g.,to improve selective targeting of allele containing thedisease-associated mutation, as long as a heteroduplex formed betweenthe oligonucleotide and target sequence is sufficiently stable towithstand the action of cellular nucleases and other modes ofdegradation which may occur in vivo. Hence, certain oligonucleotides mayhave about or at least about 70% sequence complementarity, e.g., 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence complementarity, between the oligonucleotide andthe target sequence. Oligonucleotide backbones that are less susceptibleto cleavage by nucleases are discussed herein. Mismatches, if present,are typically less destabilizing toward the end regions of the hybridduplex than in the middle. The number of mismatches allowed will dependon the length of the oligonucleotide, the percentage of G:C base pairsin the duplex, and the position of the mismatch(es) in the duplex,according to well understood principles of duplex stability.

The inhibitory nucleic acid molecule can be prepared, for example, bychemical synthesis, in vitro transcription, or digestion of long dsRNAby Rnase III or Dicer. These can be introduced into cells bytransfection, electroporation, or other methods known in the art. SeeHannon, G J, 2002, RNA Interference, Nature 418: 244-251; Bernstein E etal., 2002, The rest is silence. RNA 7: 1509-1521; Hutvagner G et al.,RNAi: Nature abhors a double-strand. Curr. Opin. Genetics & Development12: 225-232; Brummelkamp, 2002, A system for stable expression of shortinterfering RNAs in mammalian cells. Science 296: 550-553; Lee N S,Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, and Rossi J.(2002). Expression of small interfering RNAs targeted against HIV-1 revtranscripts in human cells. Nature Biotechnol. 20:500-505; Miyagishi M,and Taira K. (2002). U6-promoter-driven siRNAs with four uridine 3′overhangs efficiently suppress targeted gene expression in mammaliancells. Nature Biotechnol. 20:497-500; Paddison P J, Caudy A A, BernsteinE, Hannon G J, and Conklin D S. (2002). Short hairpin RNAs (shRNAs)induce sequence-specific silencing in mammalian cells. Genes & Dev.16:948-958; Paul C P, Good P D, Winer I, and Engelke D R. (2002).Effective expression of small interfering RNA in human cells. NatureBiotechnol. 20:505-508; Sui G, Soohoo C, Affar E-B, Gay F, Shi Y,Forrester W C, and Shi Y. (2002). A DNA vector-based RNAi technology tosuppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA99(6):5515-5520; Yu J-Y, DeRuiter S L, and Turner D L. (2002). RNAinterference by expression of short-interfering RNAs and hairpin RNAs inmammalian cells. Proc. Natl. Acad. Sci. USA 99(9):6047-6052.

In the present methods, the inhibitory nucleic acid molecule can beadministered to the subject, for example, as naked nucleic acid, incombination with a delivery reagent, and/or as a nucleic acid comprisingsequences that express an interfering nucleic acid molecule. In someembodiments the nucleic acid comprising sequences that express theinterfering nucleic acid molecules are delivered within vectors, e.g.plasmid, viral and bacterial vectors. Any nucleic acid delivery methodknown in the art can be used in the methods described herein. Suitabledelivery reagents include, but are not limited to, e.g., the MirusTransit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin;polycations (e.g., polylysine), atelocollagen, nanoplexes and liposomes.The use of atelocollagen as a delivery vehicle for nucleic acidmolecules is described in Minakuchi et al. Nucleic Acids Res.,32(13):e109 (2004); Hanai et al. Ann NY Acad Sci., 1082:9-17 (2006); andKawata et al. Mol Cancer Ther., 7(9):2904-12 (2008); each of which isincorporated herein in their entirety. Exemplary interfering nucleicacid delivery systems are provided in U.S. Pat. Nos. 8,283,461,8,313,772, 8,501,930. 8,426,554, 8,268,798 and 8,324,366, each of whichis hereby incorporated by reference in its entirety.

In some embodiments, the immunotherapy agent is an antibody or antigenbinding fragment thereof that, for example, binds to a cancer-associatedantigen. Examples of cancer-associated antigens include, but are notlimited to, adipophilin, AIM-2, ALDH1A1, alpha-actinin-4,alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABLfusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA,carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27,CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2,cyclin D1, Cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, Elongationfactor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen(“ETA”), ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-ITD, FN1,G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV,gp100/Pme117, GPNMB, HAUS3, Hepsin, HER-2/neu, HERV-K-MEL, HLA-A11,HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxylesterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1also known as CCDC110, LAGE-1, LDLR-fucosyltransferaseAS fusion protein,Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2,MATN, MC1R, MCSP, mdm-2, ME1, Melan-A/MART-1, Meloe, Midkine, MMP-2,MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I,N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1/LAGE-2, OA1, OGT, OS-9,P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein,polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA,PTPRK, RAB38/NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE,secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1,survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase,TGF-betaRII, TPBG, TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2,TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-1b/GAGED2a.In some embodiments, the antigen is a neo-antigen.

In some embodiments, the immunotherapy agent is a cancer vaccine and/ora component of a cancer vaccine (e.g., an antigenic peptide and/orprotein). The cancer vaccine can be a protein vaccine, a nucleic acidvaccine or a combination thereof. For example, in some embodiments, thecancer vaccine comprises a polypeptide comprising an epitope of acancer-associated antigen. In some embodiments, the cancer vaccinecomprises a nucleic acid (e.g., DNA or RNA, such as mRNA) that encodesan epitope of a cancer-associated antigen. In some embodiments, thenucleic acid is a vector (e.g., a bacterial vector, viral vector).Examples of bacterial vectors include, but are not limited to,Mycobacterium bovis (BCG), Salmonella Typhimurium ssp., Salmonella Typhissp., Clostridium sp. spores, Escherichia coli Nissle 1917, Escherichiacoli K-12/LLO, Listeria monocytogenes, and Shigella flexneri. Examplesof viral vectors include, but are not limited to, vaccinia, adenovirus,RNA viruses, and replication-defective avipox, replication-defectivefowlpox, replication-defective canarypox, replication-defective MVA andreplication-defective adenovirus.

In some embodiments, the cancer immunotherapy comprises administrationof an antigen presenting cell (APC) primed with a cancer-specificantigen. In some embodiments, the APC is a dendritic cell, a macrophageor a B cell.

Examples of cancer-associated antigens include, but are not limited to,adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”),ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2,beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”),CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP,COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, Cyclin-A1, dek-can fusionprotein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM,EphA3, epithelial tumor antigen (“ETA”), ETV6-AML1 fusion protein, EZH2,FGF5, FLT3-ITD, FN1, G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7,glypican-3, GnTV, gp100/Pmel 17, GPNMB, HAUS3, Hepsin, HER-2/neu,HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3,IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A,KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1,LDLR-fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-A1,MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9,MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP,mdm-2, ME1, Melan-A/MART-1, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC,mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A,neo-PAP, NFYC, NY-BR-1, NY-ESO-1/LAGE-2, OA1, OGT, OS-9, P polypeptide,p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelialmucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38/NY-MEL-1,RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernin 1, SIRT2,SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or-SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-betaRII, TPBG,TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2, TRP2-INT2,tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-1b/GAGED2a. In someembodiments, the antigen is a neo-antigen.

In some embodiments, the cancer immunotherapy comprises administrationof a cancer-specific chimeric antigen receptor (CAR). In someembodiments, the CAR is administered on the surface of a T cell. In someembodiments, the CAR binds specifically to a cancer-associated antigen.

In some embodiments, the cancer immunotherapy comprises administrationof a cancer-specific T cell to the subject. In some embodiments, the Tcell is a CD4⁺ T cell. In some embodiments, the CD4⁺ T cell is a T_(H)1T cell, a T_(H)2 T cell or a T_(H)17 T cell. In some embodiments, the Tcell expresses a T cell receptor specific for a cancer-associatedantigen.

In some embodiments, the cancer vaccine is administered with anadjuvant. Examples of adjuvants include, but are not limited to, animmune modulatory protein, Adjuvant 65, α-GalCer, aluminum phosphate,aluminum hydroxide, calcium phosphate, β-Glucan Peptide, CpG DNA,GPI-0100, lipid A, lipopolysaccharide, Lipovant, Montanide,N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A and trehalosedimycolate.

In some embodiments, the immunotherapy agent is an immune modulatingprotein to the subject. In some embodiments, the immune modulatoryprotein is a cytokine. Examples of immune modulating proteins include,but are not limited to, B lymphocyte chemoattractant (“BLC”), C-C motifchemokine 11 (“Eotaxin-1”), Eosinophil chemotactic protein 2(“Eotaxin-2”), Granulocyte colony-stimulating factor (“G-CSF”),Granulocyte macrophage colony-stimulating factor (“GM-CSF”), 1-309,Intercellular Adhesion Molecule 1 (“ICAM-1”), Interferon gamma(“IFN-gamma”), Interlukin-1 alpha (“IL-1 alpha”), Interlukin-1 beta(“IL-1 beta”), Interleukin 1 receptor antagonist (“IL-1 ra”),Interleukin-2 (“IL-2”), Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”),Interleukin-6 (“IL-6”), Interleukin-6 soluble receptor (“IL-6 sR”),Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin-10(“IL-10”), Interleukin-11 (“IL-11”), Subunit beta of Interleukin-12(“IL-12 p40” or “IL-12 p70”), Interleukin-13 (“IL-13”), Interleukin-15(“IL-15”), Interleukin-16 (“IL-16”), Interleukin-17 (“IL-17”), Chemokine(C-C motif) Ligand 2 (“MCP-1”), Macrophage colony-stimulating factor(“M-CSF”), Monokine induced by gamma interferon (“MIG”), Chemokine (C-Cmotif) ligand 2 (“MIP-1 alpha”), Chemokine (C-C motif) ligand 4 (“MIP-1beta”), Macrophage inflammatory protein-1-delta (“MIP-1 delta”),Platelet-derived growth factor subunit B (“PDGF-BB”), Chemokine (C-Cmotif) ligand 5, Regulated on Activation, Normal T cell Expressed andSecreted (“RANTES”), TIMP metallopeptidase inhibitor 1 (“TIMP-1”), TIMPmetallopeptidase inhibitor 2 (“TIMP-2”), Tumor necrosis factor,lymphotoxin-alpha (“TNF alpha”), Tumor necrosis factor, lymphotoxin-beta(“TNF beta”), Soluble TNF receptor type 1 (“sTNFRI”), sTNFRIIAR,Brain-derived neurotrophic factor (“BDNF”), Basic fibroblast growthfactor (“bFGF”), Bone morphogenetic protein 4 (‘BMP-4’), Bonemorphogenetic protein 5 (“BMP-5”), Bone morphogenetic protein 7(“BMP-7”), Nerve growth factor (“b-NGF”), Epidermal growth factor(“EGF”), Epidermal growth factor receptor (“EGFR”),Endocrine-gland-derived vascular endothelial growth factor (“EG-VEGF”),Fibroblast growth factor 4 (“FGF-4”), Keratinocyte growth factor(“FGF-7”), Growth differentiation factor 15 (“GDF-15”), Glialcell-derived neurotrophic factor (“GDNF”), Growth Hormone,Heparin-binding EGF-like growth factor (“HB-EGF”), Hepatocyte growthfactor (“HGF”), Insulin-like growth factor binding protein 1(“IGFBP-1”), Insulin-like growth factor binding protein 2 (“IGFBP-2”),Insulin-like growth factor binding protein 3 (“IGFBP-3”), Insulin-likegrowth factor binding protein 4 (“IGFBP-4”), Insulin-like growth factorbinding protein 6 (“IGFBP-6”), Insulin-like growth factor 1 (“IGF-1”),Insulin, Macrophage colony-stimulating factor (“M-CSF R”), Nerve growthfactor receptor (“NGF R”), Neurotrophin-3 (“NT-3”), Neurotrophin-4(“NT-4”), Osteoclastogenesis inhibitory factor (“Osteoprotegerin”),Platelet-derived growth factor receptors (“PDGF-AA”),Phosphatidylinositol-glycan biosynthesis (“PIGF”), Skp, Cullin, F-boxcontaining complex (“SCF”), Stem cell factor receptor (“SCF R”),Transforming growth factor alpha (“TGFalpha”), Transforming growthfactor beta-1 (“TGF beta 1”), Transforming growth factor beta-3 (“TGFbeta 3”), Vascular endothelial growth factor (“VEGF”), Vascularendothelial growth factor receptor 2 (“VEGFR2”), Vascular endothelialgrowth factor receptor 3 (“VEGFR3”), VEGF-D 6Ckine, Tyrosine-proteinkinase receptor UFO (“Axl”), Betacellulin (“BTC”), Mucosae-associatedepithelial chemokine (“CCL28”), Chemokine (C-C motif) ligand 27(“CTACK”), Chemokine (C-X-C motif) ligand 16 (“CXCL16”), C-X-C motifchemokine 5 (“ENA-78”), Chemokine (C-C motif) ligand 26 (“Eotaxin-3”),Granulocyte chemotactic protein 2 (“GCP-2”), GRO, Chemokine (C-C motif)ligand 14 (“HCC-1”), Chemokine (C-C motif) ligand 16 (“HCC-4”),Interleukin-9 (“IL-9”), Interleukin-17 F (“IL-17F”),Interleukin-18-binding protein (“IL-18 BPa”), Interleukin-28 A(“IL-28A”), Interleukin 29 (“IL-29”), Interleukin 31 (“IL-31”), C-X-Cmotif chemokine 10 (“IP-10”), Chemokine receptor CXCR3 (“I-TAC”),Leukemia inhibitory factor (“LIF”), Light, Chemokine (C motif) ligand(“Lymphotactin”), Monocyte chemoattractant protein 2 (“MCP-2”), Monocytechemoattractant protein 3 (“MCP-3”), Monocyte chemoattractant protein 4(“MCP-4”), Macrophage-derived chemokine (“MDC”), Macrophage migrationinhibitory factor (“MIF”), Chemokine (C-C motif) ligand 20 (“MIP-3alpha”), C-C motif chemokine 19 (“MIP-3 beta”), Chemokine (C-C motif)ligand 23 (“MPIF-1”), Macrophage stimulating protein alpha chain(“MSPalpha”), Nucleosome assembly protein 1-like 4 (“NAP-2”), Secretedphosphoprotein 1 (“Osteopontin”), Pulmonary and activation-regulatedcytokine (“PARC”), Platelet factor 4 (“PF4”), Stroma cell-derivedfactor-1 alpha (“SDF-1 alpha”), Chemokine (C-C motif) ligand 17(“TARC”), Thymus-expressed chemokine (“TECK”), Thymic stromallymphopoietin (“TSLP 4-IBB”), CD 166 antigen (“ALCAM”), Cluster ofDifferentiation 80 (“B7-1”), Tumor necrosis factor receptor superfamilymember 17 (“BCMA”), Cluster of Differentiation 14 (“CD14”), Cluster ofDifferentiation 30 (“CD30”), Cluster of Differentiation 40 (“CD40Ligand”), Carcinoembryonic antigen-related cell adhesion molecule 1(biliary glycoprotein) (“CEACAM-1”), Death Receptor 6 (“DR6”),Deoxythymidine kinase (“Dtk”), Type 1 membrane glycoprotein(“Endoglin”), Receptor tyrosine-protein kinase erbB-3 (“ErbB3”),Endothelial-leukocyte adhesion molecule 1 (“E-Selectin”), Apoptosisantigen 1 (“Fas”), Fms-like tyrosine kinase 3 (“Flt-3L”), Tumor necrosisfactor receptor superfamily member 1 (“GITR”), Tumor necrosis factorreceptor superfamily member 14 (“HVEM”), Intercellular adhesion molecule3 (“ICAM-3”), IL-1 R4, IL-1 RI, IL-10 Rbeta, IL-17R, IL-2Rgamma, IL-21R,Lysosome membrane protein 2 (“LIMPII”), Neutrophil gelatinase-associatedlipocalin (“Lipocalin-2”), CD62L (“L-Selectin”), Lymphatic endothelium(“LYVE-1”), MHC class I polypeptide-related sequence A (“MICA”), MHCclass I polypeptide-related sequence B (“MICB”), NRG1-beta1, Beta-typeplatelet-derived growth factor receptor (“PDGF Rbeta”), Plateletendothelial cell adhesion molecule (“PECAM-1”), RAGE, Hepatitis A viruscellular receptor 1 (“TIM-1”), Tumor necrosis factor receptorsuperfamily member IOC (“TRAIL R3”), Trappin protein transglutaminasebinding domain (“Trappin-2”), Urokinase receptor (“uPAR”), Vascular celladhesion protein 1 (“VCAM-1”), XEDARActivin A, Agouti-related protein(“AgRP”), Ribonuclease 5 (“Angiogenin”), Angiopoietin 1, Angiostatin,Catheprin S, CD40, Cryptic family protein D3 (“Cripto-1”), DAN,Dickkopf-related protein 1 (“DKK-1”), E-Cadherin, Epithelial celladhesion molecule (“EpCAM”), Fas Ligand (FasL or CD95L), Fcg RIIB/C,FoUistatin, Galectin-7, Intercellular adhesion molecule 2 (“ICAM-2”),IL-13 R1, IL-13R2, IL-17B, IL-2 Ra, IL-2 Rb, IL-23, LAP, Neuronal celladhesion molecule (“NrCAM”), Plasminogen activator inhibitor-1(“PAI-1”), Platelet derived growth factor receptors (“PDGF-AB”),Resistin, stromal cell-derived factor 1 (“SDF-1 beta”), sgp130, Secretedfrizzled-related protein 2 (“ShhN”), Sialic acid-bindingimmunoglobulin-type lectins (“Siglec-5”), ST2, Transforming growthfactor-beta 2 (“TGF beta 2”), Tie-2, Thrombopoietin (“TPO”), Tumornecrosis factor receptor superfamily member 10D (“TRAIL R4”), Triggeringreceptor expressed on myeloid cells 1 (“TREM-1”), Vascular endothelialgrowth factor C (“VEGF-C”), VEGFR1Adiponectin, Adipsin (“AND”),Alpha-fetoprotein (“AFP”), Angiopoietin-like 4 (“ANGPTL4”),Beta-2-microglobulin (“B2M”), Basal cell adhesion molecule (“BCAM”),Carbohydrate antigen 125 (“CA125”), Cancer Antigen 15-3 (“CA15-3”),Carcinoembryonic antigen (“CEA”), cAMP receptor protein (“CRP”), HumanEpidermal Growth Factor Receptor 2 (“ErbB2”), Follistatin,Follicle-stimulating hormone (“FSH”), Chemokine (C-X-C motif) ligand 1(“GRO alpha”), human chorionic gonadotropin (“beta HCG”), Insulin-likegrowth factor 1 receptor (“IGF-1 sR”), IL-1 sRII, IL-3, IL-18 Rb, IL-21,Leptin, Matrix metalloproteinase-1 (“MMP-1”), Matrix metalloproteinase-2(“MMP-2”), Matrix metalloproteinase-3 (“MMP-3”), Matrixmetalloproteinase-8 (“MMP-8”), Matrix metalloproteinase-9 (“MMP-9”),Matrix metalloproteinase-10 (“MMP-10”), Matrix metalloproteinase-13(“MMP-13”), Neural Cell Adhesion Molecule (“NCAM-1”), Entactin(“Nidogen-1”), Neuron specific enolase (“NSE”), Oncostatin M (“OSM”),Procalcitonin, Prolactin, Prostate specific antigen (“PSA”), Sialicacid-binding Ig-like lectin 9 (“Siglec-9”), ADAM 17 endopeptidase(“TACE”), Thyroglobulin, Metalloproteinase inhibitor 4 (“TIMP-4”),TSH2B4, Disintegrin and metalloproteinase domain-containing protein 9(“ADAM-9”), Angiopoietin 2, Tumor necrosis factor ligand superfamilymember 13/Acidic leucine-rich nuclear phosphoprotein 32 family member B(“APRIL”), Bone morphogenetic protein 2 (“BMP-2”), Bone morphogeneticprotein 9 (“BMP-9”), Complement component 5a (“C5a”), Cathepsin L,CD200, CD97, Chemerin, Tumor necrosis factor receptor superfamily member6B (“DcR3”), Fatty acid-binding protein 2 (“FABP2”), Fibroblastactivation protein, alpha (“FAP”), Fibroblast growth factor 19(“FGF-19”), Galectin-3, Hepatocyte growth factor receptor (“HGF R”),IFN-gammalpha/beta R2, Insulin-like growth factor 2 (“IGF-2”),Insulin-like growth factor 2 receptor (“IGF-2 R”), Interleukin-1receptor 6 (“IL-1R6”), Interleukin 24 (“IL-24”), Interleukin 33(“IL-33”, Kallikrein 14, Asparaginyl endopeptidase (“Legumain”),Oxidized low-density lipoprotein receptor 1 (“LOX-1”), Mannose-bindinglectin (“MBL”), Neprilysin (“NEP”), Notch homolog 1,translocation-associated (Drosophila) (“Notch-1”), Nephroblastomaoverexpressed (“NOV”), Osteoactivin, Programmed cell death protein 1(“PD-1”), N-acetylmuramoyl-L-alanine amidase (“PGRP-5”), Serpin A4,Secreted frizzled related protein 3 (“sFRP-3”), Thrombomodulin, Tolllikereceptor 2 (“TLR2”), Tumor necrosis factor receptor superfamily member10A (“TRAIL R1”), Transferrin (“TRF”), WIF-1ACE-2, Albumin, AMICA,Angiopoietin 4, B-cell activating factor (“BAFF”), Carbohydrate antigen19-9 (“CA19-9”), CD 163, Clusterin, CRT AM, Chemokine (C-X-C motif)ligand 14 (“CXCL14”), Cystatin C, Decorin (“DCN”), Dickkopf-relatedprotein 3 (“Dkk-3”), Delta-like protein 1 (“DLL1”), Fetuin A,Heparin-binding growth factor 1 (“aFGF”), Folate receptor alpha(“FOLR1”), Furin, GPCR-associated sorting protein 1 (“GASP-1”),GPCR-associated sorting protein 2 (“GASP-2”), Granulocytecolony-stimulating factor receptor (“GCSF R”), Serine protease hepsin(“HAI-2”), Interleukin-17B Receptor (“IL-17B R”), Interleukin 27(“IL-27”), Lymphocyte-activation gene 3 (“LAG-3”), Apolipoprotein A-V(“LDL R”), Pepsinogen I, Retinol binding protein 4 (“RBP4”), SOST,Heparan sulfate proteoglycan (“Syndecan-1”), Tumor necrosis factorreceptor superfamily member 13B (“TACI”), Tissue factor pathwayinhibitor (“TFPI”), TSP-1, Tumor necrosis factor receptor superfamily,member 10b (“TRAIL R2”), TRANCE, Troponin I, Urokinase PlasminogenActivator (“uPA”), Cadherin 5, type 2 or VE-cadherin (vascularendothelial) also known as CD144 (“VE-Cadherin”),WNT1-inducible-signaling pathway protein 1 (“WISP-1”), and ReceptorActivator of Nuclear Factor κ B (“RANK”).

In some embodiments, the cancer therapeutic is a radioactive moiety thatcomprises a radionuclide. Exemplary radionuclides include, but are notlimited to Cr-51, Cs-131, Ce-134, Se-75, Ru-97, I-125, Eu-149, Os-189m,Sb-119, I-123, Ho-161, Sb-117, Ce-139, In-111, Rh-103m, Ga-67, T1-201,Pd-103, Au-195, Hg-197, Sr-87m, Pt-191, P-33, Er-169, Ru-103, Yb-169,Au-199, Sn-121, Tm-167, Yb-175, In-113m, Sn-113, Lu-177, Rh-105,Sn-117m, Cu-67, Sc-47, Pt-195m, Ce-141, I-131, Tb-161, As-77, Pt-197,Sm-153, Gd-159, Tm-173, Pr-143, Au-198, Tm-170, Re-186, Ag-111, Pd-109,Ga-73, Dy-165, Pm-149, Sn-123, Sr-89, Ho-166, P-32, Re-188, Pr-142,Ir-194, In-114m/In-114, and Y-90.

In some embodiments, the cancer therapeutic is an angiogenesis inhibitorto the subject. Examples of such angiogenesis inhibitors include, butare not limited to Bevacizumab (Avastin®), Ziv-aflibercept (Zaltrap®),Sorafenib (Nexavar®), Sunitinib (Sutent®), Pazopanib (Votrient®),Regorafenib (Stivarga®), and Cabozantinib (Cometrig™).

In some embodiments, the cancer therapeutic is an antibiotic. Forexample, if the presence of a cancer-associated bacteria and/or acancer-associated microbiome profile is detected according to themethods provided herein, antibiotics can be administered to eliminatethe cancer-associated bacteria from the subject. “Antibiotics” broadlyrefers to compounds capable of inhibiting or preventing a bacterialinfection. Antibiotics can be classified in a number of ways, includingtheir use for specific infections, their mechanism of action, theirbioavailability, or their spectrum of target microbe (e.g.,Gram-negative vs. Gram-positive bacteria, aerobic vs. anaerobicbacteria, etc.) and these may be used to kill specific bacteria inspecific areas of the host (“niches”) (Leekha, et al 2011. GeneralPrinciples of Antimicrobial Therapy. Mayo Clin Proc. 86(2): 156-167). Incertain embodiments, antibiotics can be used to selectively targetbacteria of a specific niche. In some embodiments, antibiotics known totreat a particular infection that includes a cancer niche may be used totarget cancer-associated microbes, including cancer-associated bacteriain that niche. In other embodiments, antibiotics are administered afterthe bacterial treatment. In some embodiments, antibiotics areadministered after the bacterial treatment to remove the engraftment.

In some aspects, antibiotics can be selected based on their bactericidalor bacteriostatic properties. Bactericidal antibiotics includemechanisms of action that disrupt the cell wall (e.g., β-lactams), thecell membrane (e.g., daptomycin), or bacterial DNA (e.g.,fluoroquinolones). Bacteriostatic agents inhibit bacterial replicationand include sulfonamides, tetracyclines, and macrolides, and act byinhibiting protein synthesis. Furthermore, while some drugs can bebactericidal in certain organisms and bacteriostatic in others, knowingthe target organism allows one skilled in the art to select anantibiotic with the appropriate properties. In certain treatmentconditions, bacteriostatic antibiotics inhibit the activity ofbactericidal antibiotics. Thus, in certain embodiments, bactericidal andbacteriostatic antibiotics are not combined.

Antibiotics include, but are not limited to aminoglycosides, ansamycins,carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides,lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones,penicillins, polypeptide antibiotics, quinolones, fluoroquinolone,sulfonamides, tetracyclines, and anti-mycobacterial compounds, andcombinations thereof.

Aminoglycosides include, but are not limited to Amikacin, Gentamicin,Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, andSpectinomycin. Aminoglycosides are effective, e.g., againstGram-negative bacteria, such as Escherichia coli, Klebsiella,Pseudomonas aeruginosa, and Francisella tularensis, and against certainaerobic bacteria but less effective against obligate/facultativeanaerobes. Aminoglycosides are believed to bind to the bacterial 30S or50S ribosomal subunit thereby inhibiting bacterial protein synthesis.

Ansamycins include, but are not limited to, Geldanamycin, Herbimycin,Rifamycin, and Streptovaricin. Geldanamycin and Herbimycin are believedto inhibit or alter the function of Heat Shock Protein 90.

Carbacephems include, but are not limited to, Loracarbef. Carbacephemsare believed to inhibit bacterial cell wall synthesis.

Carbapenems include, but are not limited to, Ertapenem, Doripenem,Imipenem/Cilastatin, and Meropenem. Carbapenems are bactericidal forboth Gram-positive and Gram-negative bacteria as broad-spectrumantibiotics. Carbapenems are believed to inhibit bacterial cell wallsynthesis.

Cephalosporins include, but are not limited to, Cefadroxil, Cefazolin,Cefalotin, Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin,Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone,Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime,Ceftriaxone, Cefepime, Ceftaroline fosamil, and Ceftobiprole. SelectedCephalosporins are effective, e.g., against Gram-negative bacteria andagainst Gram-positive bacteria, including Pseudomonas, certainCephalosporins are effective against methicillin-resistantStaphylococcus aureus (MRSA). Cephalosporins are believed to inhibitbacterial cell wall synthesis by disrupting synthesis of thepeptidoglycan layer of bacterial cell walls.

Glycopeptides include, but are not limited to, Teicoplanin, Vancomycin,and Telavancin. Glycopeptides are effective, e.g., against aerobic andanaerobic Gram-positive bacteria including MRSA and Clostridiumdifficile. Glycopeptides are believed to inhibit bacterial cell wallsynthesis by disrupting synthesis of the peptidoglycan layer ofbacterial cell walls.

Lincosamides include, but are not limited to, Clindamycin andLincomycin. Lincosamides are effective, e.g., against anaerobicbacteria, as well as Staphylococcus, and Streptococcus. Lincosamides arebelieved to bind to the bacterial 50S ribosomal subunit therebyinhibiting bacterial protein synthesis.

Lipopeptides include, but are not limited to, Daptomycin. Lipopeptidesare effective, e.g., against Gram-positive bacteria. Lipopeptides arebelieved to bind to the bacterial membrane and cause rapiddepolarization.

Macrolides include, but are not limited to, Azithromycin,Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin,Troleandomycin, Telithromycin, and Spiramycin. Macrolides are effective,e.g., against Streptococcus and Mycoplasma. Macrolides are believed tobind to the bacterial or 50S ribosomal subunit, thereby inhibitingbacterial protein synthesis.

Monobactams include, but are not limited to, Aztreonam. Monobactams areeffective, e.g., against Gram-negative bacteria. Monobactams arebelieved to inhibit bacterial cell wall synthesis by disruptingsynthesis of the peptidoglycan layer of bacterial cell walls.

Nitrofurans include, but are not limited to, Furazolidone andNitrofurantoin.

Oxazolidonones include, but are not limited to, Linezolid, Posizolid,Radezolid, and Torezolid. Oxazolidonones are believed to be proteinsynthesis inhibitors.

Penicillins include, but are not limited to, Amoxicillin, Ampicillin,Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin,Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, PenicillinV, Piperacillin, Temocillin and Ticarcillin. Penicillins are effective,e.g., against Gram-positive bacteria, facultative anaerobes, e.g.,Streptococcus, Borrelia, and Treponema. Penicillins are believed toinhibit bacterial cell wall synthesis by disrupting synthesis of thepeptidoglycan layer of bacterial cell walls.

Penicillin combinations include, but are not limited to,Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam,and Ticarcillin/clavulanate.

Polypeptide antibiotics include, but are not limited to, Bacitracin,Colistin, and Polymyxin B and E. Polypeptide Antibiotics are effective,e.g., against Gram-negative bacteria. Certain polypeptide antibioticsare believed to inhibit isoprenyl pyrophosphate involved in synthesis ofthe peptidoglycan layer of bacterial cell walls, while othersdestabilize the bacterial outer membrane by displacing bacterialcounter-ions.

Quinolones and Fluoroquinolone include, but are not limited to,Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,Trovafloxacin, Grepafloxacin, Sparfloxacin, and Temafloxacin.Quinolones/Fluoroquinolone are effective, e.g., against Streptococcusand Neisseria. Quinolones/Fluoroquinolone are believed to inhibit thebacterial DNA gyrase or topoisomerase IV, thereby inhibiting DNAreplication and transcription.

Sulfonamides include, but are not limited to, Mafenide, Sulfacetamide,Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole,Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole,Trimethoprim-Sulfamethoxazole (Co-trimoxazole), andSulfonamidochrysoidine. Sulfonamides are believed to inhibit folatesynthesis by competitive inhibition of dihydropteroate synthetase,thereby inhibiting nucleic acid synthesis.

Tetracyclines include, but are not limited to, Demeclocycline,Doxycycline, Minocycline, Oxytetracycline, and Tetracycline.Tetracyclines are effective, e.g., against Gram-negative bacteria.Tetracyclines are believed to bind to the bacterial 30S ribosomalsubunit thereby inhibiting bacterial protein synthesis.

Anti-mycobacterial compounds include, but are not limited to,Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide,Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, andStreptomycin.

Suitable antibiotics also include arsphenamine, chloramphenicol,fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin,quinupristin/dalfopristin, tigecycline, tinidazole, trimethoprimamoxicillin/clavulanate, ampicillin/sulbactam, amphomycin ristocetin,azithromycin, bacitracin, buforin II, carbomycin, cecropin Pl,clarithromycin, erythromycins, furazolidone, fusidic acid, Na fusidate,gramicidin, imipenem, indolicidin, josamycin, magainan II,metronidazole, nitroimidazoles, mikamycin, mutacin B-Ny266, mutacinB-JH1 140, mutacin J-T8, nisin, nisin A, novobiocin, oleandomycin,ostreogrycin, piperacillin/tazobactam, pristinamycin, ramoplanin,ranalexin, reuterin, rifaximin, rosamicin, rosaramicin, spectinomycin,spiramycin, staphylomycin, streptogramin, streptogramin A, synergistin,taurolidine, teicoplanin, telithromycin, ticarcillin/clavulanic acid,triacetyloleandomycin, tylosin, tyrocidin, tyrothricin, vancomycin,vemamycin, and virginiamycin.

In some embodiments, the the cancer therapy comprises administering atherapeutic bacteria and/or a therapeutic combination of bacteria to thesubject so a healthy microbiome can be reconstituted in the subject. Insome embodiments, the therapeutic bacteria is a non-cancer-associatedbacteria. In some embodiments the therapeutic bacteria is a probioticbacteria.

Cancer

In some embodiments, the methods and compositions described hereinrelate to the treatment of cancer. Examples of cancers that may treatedby methods described herein include, but are not limited to,hematological malignancy, acute nonlymphocytic leukemia, chroniclymphocytic leukemia, acute granulocytic leukemia, chronic granulocyticleukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemicleukemia, a leukocythemic leukemia, basophilic leukemia, blast cellleukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis,embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, undifferentiated cell leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma,carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidalcell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinomavillosum, carcinoma gigantocellulare, glandular carcinoma, granulosacell carcinoma, hair-matrix carcinoma, hematoid carcinoma,hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma,hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma insitu, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, carcinoma scroti, chondrosarcoma, fibrosarcoma,lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrialsarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblasticsarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma,liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoidsarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms'tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathicmultiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of Bcells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma,Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma,malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocyticsarcoma, rhabdosarcoma, serocystic sarcoma, synovial sarcoma,telangiectaltic sarcoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma,multiple myeloma, neuroblastoma, bladder cancer, breast cancer, ovariancancer, lung cancer, colorectal cancer, rhabdomyosarcoma, primarythrombocytosis, primary macroglobulinemia, small-cell lung tumors,primary brain tumors, stomach cancer, colon cancer, malignant pancreaticinsulanoma, malignant carcinoid, premalignant skin lesions, testicularcancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,genitourinary tract cancer, malignant hypercalcemia, cervical cancer,endometrial cancer, adrenal cortical cancer, Harding-Passey melanoma,juvenile melanoma, lentigo maligna melanoma, malignant melanoma,acral-lentiginous melanoma, amelanotic melanoma, benign juvenilemelanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungalmelanoma, superficial spreading melanoma, plasmacytoma, colorectalcancer, rectal cancer.

In some embodiments, the methods and compositions provided herein relateto the treatment of a leukemia. The term “leukemia” is meant broadlyprogressive, malignant diseases of the hematopoietic organs/systems andis generally characterized by a distorted proliferation and developmentof leukocytes and their precursors in the blood and bone marrow.Non-limiting examples of leukemia diseases include, acute nonlymphocyticleukemia, chronic lymphocytic leukemia, acute granulocytic leukemia,chronic granulocytic leukemia, acute promyelocytic leukemia, adultT-cell leukemia, aleukemic leukemia, a leukocythemic leukemia,basophilic leukemia, blast cell leukemia, bovine leukemia, chronicmyelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilicleukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia,stem cell leukemia, subleukemic leukemia, undifferentiated cellleukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblasticleukemia, histiocytic leukemia, stem cell leukemia, acute monocyticleukemia, leukopenic leukemia, lymphatic leukemia, lymphoblasticleukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoidleukemia, lymphosarcoma cell leukemia, mast cell leukemia,megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia,myeloblastic leukemia, myelocytic leukemia, myeloid granulocyticleukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cellleukemia, plasmacytic leukemia, and promyelocytic leukemia.

In some embodiments, the methods and compositions provided herein relateto the treatment of a carcinoma. The term “carcinoma” refers to amalignant growth made up of epithelial cells tending to infiltrate thesurrounding tissues, and/or resist physiological and non-physiologicalcell death signals and gives rise to metastases. Non-limiting exemplarytypes of carcinomas include, acinar carcinoma, acinous carcinoma,adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum,carcinoma of adrenal cortex, alveolar carcinoma, alveolar cellcarcinoma, basal cell carcinoma, carcinoma basocellulare, basaloidcarcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma,comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cellcarcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiennoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma,signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma,solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma,carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma,string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes,transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma,verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypernephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, merkel cell carcinoma, and salivary gland carcinoma.

In some embodiments, the methods and compositions provided herein relateto the treatment of a sarcoma. The term “sarcoma” generally refers to atumor which is made up of a substance like the embryonic connectivetissue and is generally composed of closely packed cells embedded in afibrillar, heterogeneous, or homogeneous substance. Sarcomas include,but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma,melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromalsarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giantcell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolarsoft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloromasarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma,granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmentedhemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma,immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma,Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymomasarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma,serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

Additional exemplary neoplasias that can be treated using the methodsand compositions described herein include Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer,ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, small-cell lung tumors, primary brain tumors,stomach cancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,and adrenal cortical cancer.

In some embodiments, the cancer treated is a melanoma. The term“melanoma” is taken to mean a tumor arising from the melanocytic systemof the skin and other organs. Non-limiting examples of melanomas areHarding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma,malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma,benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodularmelanoma subungal melanoma, and superficial spreading melanoma.

Particular categories of tumors that can be treated using methods andcompositions described herein include lymphoproliferative disorders,breast cancer, ovarian cancer, prostate cancer, cervical cancer,endometrial cancer, bone cancer, liver cancer, stomach cancer, coloncancer, colorectal cancer, pancreatic cancer, cancer of the thyroid,head and neck cancer, cancer of the central nervous system, cancer ofthe peripheral nervous system, skin cancer, kidney cancer, as well asmetastases of all the above. Particular types of tumors includehepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma,esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma,rhabdotheliosarcoma, invasive ductal carcinoma, papillaryadenocarcinoma, melanoma, pulmonary squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma (well differentiated, moderatelydifferentiated, poorly differentiated or undifferentiated),bronchioloalveolar carcinoma, renal cell carcinoma, hypernephroma,hypernephroid adenocarcinoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lungcarcinoma including small cell, non-small and large cell lung carcinoma,bladder carcinoma, glioma, astrocyoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma,colon carcinoma, rectal carcinoma, hematopoietic malignancies includingall types of leukemia and lymphoma including: acute myelogenousleukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia,multiple myeloma, myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin'slymphoma.

Cancers treated in certain embodiments also include precancerouslesions, e.g., actinic keratosis (solar keratosis), moles (dysplasticnevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett'sesophagus, atrophic gastritis, dyskeratosis congenita, sideropenicdysphagia, lichen planus, oral submucous fibrosis, actinic (solar)elastosis and cervical dysplasia.

Cancers treated in some embodiments include non-cancerous or benigntumors, e.g., of endodermal, ectodermal or mesenchymal origin,including, but not limited to cholangioma, colonic polyp, adenoma,papilloma, cystadenoma, liver cell adenoma, hydatidiform mole, renaltubular adenoma, squamous cell papilloma, gastric polyp, hemangioma,osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma,rhabdomyoma, astrocytoma, nevus, meningioma, and ganglioneuroma.

Examples Example 1: Culture of Blautia massiliensis Strain A onSelective Media

Required Equipment:

Anaerobic chamber (10% H2, 5% CO2, 85% N2 atmosphere at 35-370C)Multi sample vortex100 uL pipettor

Consumables:

Sterile loopsColony spreader

Pipets

Pipet tips96-well sterile plates96-well deep-well plates

Media:

TABLE 1 Media preparation sheet. Amount Expiration Component Conc./LAdded Brand Catalog # Lot # Date 1. Fastidious — 45.7 g/L Acumedia —107852B Jul. 31, 2019 anaerobes agar (FAA)powder +Yeast   5 g/L   5 g/LFisher — 146772 August 2017 extract(granulated) bioreagents+Erythromycin to   32 ug/L 1 mL/1 L MP Biomedicals 190197 M9033 January2017 make (FAA + Ery) +Ciprofloxacin to   16 ug/L 1 mL/1 L Sigma449620050 A0355311 N/A make (FAA + Cipro) +Ampicillin to   50 ug/L 1mL/1 L Fisher Scientific 69523, 151231 N/A make BP1760-25 (FAA + Ampi)+Tetracycline to 16000 ug/L 1 mL/1 L Fisher 64755 152874 N/A make (FAA +TET) 2. GAM broth GAM broth   59 g/L HIMEDIA/Fisher M1801/BP1423 N/A N/Awith agar with1.5% agar 3. Anaerobe —   46 g/L Oxoid CM0972 N/A N/ABasal Agar.

Fastidious anaerobic agar (FAAy) (Acumedia7531A) with Antibiotics.Selective media prepared at Epiva.

These antibiotics are added to the media in order to inhibit the growthof other group of bacteria that might interfere with the isolation ofBlautia.

FAA+50 ug/ml AmpicillinFAA+16 ug/ml ErythromycinFAA+16 ug/ml Ciprofloxacin

FAA+32 ug/ml TetracyclineFastidious Anaerobe Agar is a customformulation used for the cultivation of various fastidious anaerobesfrom clinical and nonclinical specimens. Peptone provides nitrogen andvitamin sources in Fastidious Anaerobe Agar. Sodium Chloride maintainsthe osmotic balance of the medium. Soluble Starch is present to absorbany toxic metabolites. Sodium Bicarbonate increases the aerotolerance byacting as an oxygen scavenger. Sodium Pyrophosphate is a bufferingagent. Glucose is the carbon source. Sodium Pyruvate is added as anenergy source and as an oxygen scavenger for asaccharolytic cocci,including Veillonella spp. L-Cysteine HCl.H20 is a reducing agent andgrowth stimulant for anaerobes. L-Arginine is added to ensure the growthof Eubacterium lentum, and Hemin and Vitamin K are growth factorsrequired by several Bacteroides spp. Sodium Succinate improves thegrowth of Prevotella melaninogenica and Bacteroides spp.

Anaerobe Basal Agar. (Oxoid CM0972)

Anaerobe Basal Agar contains peptones, carefully selected to supportgood growth of anaerobic bacteria and yeast extract as a vitamin source.Starch is present to absorb any toxic metabolites1. Sufficient arginineis added to ensure the growth of Eubacterium, lentum, hemin and vitaminK are growth factors required by many Bacteroides species. Hemin is alsorequired by Porphyromonas species. Sodium succinate improves the growthof Prevotella, melaninogenica and Bacteroides species4. Sodium pyruvateis added as an energy source for asaccharolytic cocci such asVeillonella. It also acts similarly to catalase and degrades traces ofhydrogen peroxide, which may be produced by the action of molecularoxygen on media components5. L-cysteine hydrochloride and dithiothreitolare reducing agents, and cysteine has also been shown to stimulate thegrowth of some anaerobes

Gifu Anaerobic Medium (GAM Broth) (HIMEDIA M1801) Agar (FisherBP1423)

This is a liquid medium for anaerobic bacteria. As this medium containsthe digested serum which has hemin, serves as essential growth factorand Sodium chloride maintains osmotic equilibrium. Also contains Sodiumthioglycolate and L-Cysteine are the reducing agents added in thismedium to provide adequate anaerobiosis. Peptic digest of animal tissueand yeast extract provide nitrogen, carbon and vitamin source. Starchabsorbs the toxic metabolites produced. Anaerobic organisms requirereducing condition and an absence of dissolved oxygen in the medium.Strict anaerobes obtain its energy and intermediates through oxidationutilizing hydrogen acceptors other than oxygen.

Peptone Yeast (PY)(AS-821) with: Glycogen, N-Acetil-D-Glucosamine,Cellulobiose.

PRAS (Pre-reduced Anaerobically Sterilized) Peptone Yeast Extract BrothBased Media (PY) are enriched non-selective broth media used in thegrowth and partial identification of anaerobic bacteria. Peptone YeastExtract Broth Based Media with carbohydrate/biochemical additives aredesigned for determination of specific biochemical features of anaerobicbacteria. This biochemical determination is used to aid in thedefinitive identification of anaerobic bacteria. Peptone Yeast ExtractBroth Based Media are enriched non-selective media that are supplementedwith hemin and vitamin K1 to facilitate the recovery of the morefastidious organisms such as Prevotella, Porphyromonas and theBacteroides fragilis group. Resazurin is added as a redox indicator.PRAS biochemicals are considered to be the “gold standard” forbiochemical testing and characterization of anaerobes. With the additionof a specific biochemical, the Peptone Yeast Extract Broth Based Mediaare used to determine whether anaerobic bacteria will utilize thebiochemical or if growth is stimulated or inhibited by the biochemical.

Procedure

Step 1. Recovery of Bacteria from Frozen Stool Samples.

Frozen stool sample is transferred to the anaerobic chamber to workwith.

The sample that is left on a swab is placed onto a new sterile transportmedia for storage at −80C freezer as soon as possible to preserve thesample for further applications.

2.5 ml of sterile Phosphate Buffered Saline (PBS) is added in thetransport media (original sample) containing the rest of the stoolsample. The mixture is mixed very well, vortexed gently if required

1 ml aliquot is remove and added to a tube containing 9 mL of sterilePhosphate Buffered Saline (PBS) resulting (1:10 dilution). The mixtureis vortexed gently. The remaining sample is frozen at −80C.

1 mL from 1:10 (10⁻¹) is transferred into a 9 mL tube of PBS resultingin 1:100 (10-2). Continue the serial dilution to reach a dilution of10⁻⁵

Step 2. Enumeration of Solid Media.

The following agar plates are labeled with the subject information anddilution factor:

GAM with Agar,

Anaerobic Basal Agar,

FAA+50 ug/ml Ampicillin,FAA+32 ug/ml Erythromycin,FAA+16 ug/ml Ciprofloxacin,FAA+16 ug/ml Tetracycline,

PY-glycogen, PY-N-Acetil-D-Glucosamine, PY-Cellobiose

100 uL aliquots of dilutions 10⁻³, 10⁻⁴, 10⁻⁵ is transferred in onto thesurface of the agar plates. This results in a final dilution range of10⁻⁴, 10⁻⁵ and 10⁻⁶ CFU/ml of stool.

One of each plate is labeled NEGATIVE Control. 100 uL of sterile PBS istransferred on to surface of plate. This is to verify the sterility ofPBS, media plates and colony spreader at the time of use.

After plating, diluted samples are stored at RT.

A colony spreader is used to spread aliquot evenly across surface ofagar. Plates are allowed to seat for approximately 10 min (or until dry)to allow spread inoculum to absorb into the plates.

Inverted plates are incubated in the anaerobic chamber at 37° C. for48-72 hours (or until colonies are visible up to 5 days).

plates are checked based on colony morphology pick 3 isolated coloniesof the same morphology for PCR and re-isolate them on BHI, or CDC bloodagar plates to perform further testing and/or isolate single coloniesfrom a pure culture for PCR.

Colonies are picked using either sterile disposable loops or pipettetips, and diluted in 100 μL distilled water (easiest to do this in a96-well plate).

These suspensions are then cycled outside of the chamber, while theplates remain for downstream recovery of strains of interest.

PCR is performed using the following reaction mixture (in a 96-well PCRplate):

a-12.5 μL Go Taq Green Master Mix (Promega, cat # M7123)

b-0.2 μL 16S rDNA primer 27F  (5′ AGAGTTTGATCMTGGCTCAG 3′(SEQ ID NO: 1)) c-0.2 μL 16S rDNA primer 1492R  (5′TACGGYTACCTTGTTACGACTT 3 (SEQ ID NO: 2))d-11.1 μL nuclease free distilled water (sent with the master mix)e-1 μL diluted cell inoculumPCR protocol is as follows . . .a. 10-minute denaturation at 95° C.b. 30 seconds at 95° C.c. 30 seconds at 59° C.d. 30 seconds at 72° C.e. (repeated b-d 30 times)f. 5 minutes at 72° C.g. Hold at 4° C.

Successful PCR is confirmed on 18 reactions using gel electrophoresis.If over 60% success, 96-well plate of PCRs is sent to Genewiz forsequencing. Isolated colonies confirmed by PCR are grown in liquidculture, and cryopreserved as described above. Pictures of colonymorphology and Gram stain are taken using microscope (100×magnification). If multiple isolates are identified, bacteria are grownand stored in 96 deep-well plates.

Example 2: Blautia Massiliensis Strain A Production

Enriched media is used to grow and prepare Blautia massiliensis for invitro and in vivo use. For example, media may contain sugar, yeastextracts, peptones, trace elements, and vitamins. Alternatively, mediamay be prepared and Blautia massiliensis grown as is known in the art.Influence of fermentation time, cryoprotectant and neutralization ofcell concentrate on freeze-drying survival, storage stability, and acidand bile exposure of Blautia massiliensis cells produced withoutmilk-based ingredients,

At large scale, the media is sterilized. Sterilization may be by UltraHigh Temperature (UHT) processing. The UHT processing is performed atvery high temperature for short periods of time. The UHT range may befrom 135-180° C. For example, the medium may be sterilized from between10 to 30 seconds at 135° C.

Inoculum can be prepared in flasks or in smaller bioreactors and growthis monitored. For example, the inoculum size may be betweenapproximately 0.5 and 3% of the total bioreactor volume. Depending onthe application and need for material, bioreactor volume can be at least2 L, 10 L, 80 L, 100 L, 250 L, 1000 L, 2500 L, 5000 L, 10,000 L.

Before the inoculation, the bioreactor is prepared and ready with mediumat desired pH, temperature, and oxygen concentration. For example, pHmay be set between 4.5 and 8.0 During the fermentation the pH can becontrolled through the use of sodium hydroxide, potassium hydroxide, orammonium hydroxide. The temperature may be controlled from 25° C. to 45°C., for example at 37° C. Anaerobic conditions are created by reducingthe level of oxygen in the culture broth from around 8 mg/L to 0 mg/L.For example, nitrogen or gas mixtures (N₂, CO₂, and H₂) may be used inorder to establish anaerobic conditions. Alternatively, no gases areused and anaerobic conditions are established by cells consumingremaining oxygen from the medium. Depending on strain and inoculum size,the bioreactor fermentation time can vary. For example, fermentationtime can vary from approximately 5 hours to 48 hours.

Harvesting can be performed by continuous centrifugation. Product willbe resuspended with various excipients to a desired final concentration.Excipients can be added for cryo protection or for protection duringlyophilization. Excipients can include, but are not limited to, sucrose,trehalose, or lactose, and these may be alternatively mixed with bufferand antioxidants. Prior to lyophilization, droplets of cell pelletsmixed with excipients are submerged in liquid nitrogen.

Lyophilization of material, including live bacteria, begins with primarydrying. During the primary drying phase, the ice is removed. Here, avacuum is generated and an appropriate amount of heat is supplied to thematerial for the ice to sublime. During the secondary drying phase,product bound water molecules are removed. Here, the temperature israised higher than in the primary drying phase to break anyphysico-chemical interactions that have formed between the watermolecules and the product material. The pressure may also be loweredfurther to enhance desorption during this stage. After the freeze-dryingprocess is complete, the chamber may be filled with an inert gas, suchas nitrogen. The product may be sealed within the freeze dryer under dryconditions, preventing exposure to atmospheric water and contaminants.

Example 3: Blautia Massiliensis Strain A Fermentation Process TSBFPreparation:

TABLE 2 Group 1 components Group 1 g/L Yeast Extract 19512 OrganotechnieS.A.S. 10 Soy Peptone E110 19885 Organotechnie S.A.S 10 Soy Peptone A2SC 19649 Organotechnie S.A.S 10 Potato Peptone E210 19425 OrganotechnieS.A.S 5 Dipotassium Phosphate K₂HPO₄ 2.5 Sodium Chloride 5

Group 1 components are combined in appropriate volume of DI water,remembering to multiply 1 L by the number of liters desired. The mixtureis then autoclave for 60 minutes with a maximum volume in each containerof 2.5 L.

Group 2 components: L-Cysteine-HCl (50 g/L) and FeSO₄ (5 g/L)

Group 2 components are combined with appropriate volume of DI water. Themixture is aliquoted into 100 mL volumes in 200 mL bottles. Rubberstoppers and crimp metal cap are inserted. The mixture is thenautoclaved for 30 minutes.

Glucose

There are two (2) types of glucose stocks. One is the pre-sterilized 45%glucose solutions from Sigma Aldrich (use 11.1 ml/L). The other is the50% filter sterilized stocks made in-house (use 10.0 ml/L).

To make 50% stocks, 1 L of DI water is heated in microwave for 2.5minutes. 400 mL of heated water was stirred vigorously while slowlyadding 500 g of glucose, not allowing glucose to clump in solution.After all glucose is dissolved, solution is allowed to continue mixingwhile heating on stir plate for 5 minutes. Hot DI water is then added upto 1 L volume. The mixture is filtered sterilize using 0.2 um vacuumfilter. 5 g/L is the concentration of glucose in the final medium.

Combination: 10 mL of Group 2 is added for every liter of Group 1.Appropriate volume of glucose stock solution is then added.

Objective: To investigate the influence of pH and alternativecryoprotectants on growth and stability of Blautia massiliensis strainA. To observe the correlation between CFU and GDA as well as thevariance between these methods of analysis. GDA data are used to find anacceptable dilution that allows high resolution and comparison betweenGDA and CFU. D002 are also used to test changes to pH control PID.Finally, procedures and run-sheets are changed based upon improvementsmade during D002. Downsteam sampling is performed with 4 differentstabilizers and one control.

Procedures: The following procedures were used to setup and executeDasGip fermentation run D002. See the procedure archive for thematerials and methods associated with each listed procedure.

DasGip Setup Laboratory Procedure v0.1 Anaerobic Inoculum PreparationLaboratory Procedure v0.1 Bioreactor Inoculation Laboratory Procedurev0.1 DasGip Sampling and Analysis Laboratory Procedure v0.1 ColonyForming Units Laboratory Procedure v0.1

Optical Density and Growth Detection Assay Laboratory procedure v0.1

Methods: Select methods and parameters are described below as well asany methods not included in the procedures above and/or deviating fromthe noted procedures.

Media: Media used for fermentation and inoculum was prepared per BaseMedium “BM1” Media Prep. Formulation v0.1. The following batch(s) wasused: BM109271601. Media was added to each vessel under sterileconditions in a biosafety cabinet. pH adjustments were performed usingthe DasGip controller (10N NaOH). Media was de-gassed overnight to undermixed gas sparging (5% H2, 5% CO2, Balance N2) prior to inoculation. 10ppm of sterile Antifoam Emulsion C was added via sterile syringe afterde-gassing.

Calibrations: Calibrations of pH and RD probes were performed prior toautoclaving of the vessels. A clean in place procedure was performed onpump “B” prior to connecting base lines to each vessel. Calibrationvalues and probe serial numbers are listed below.

TABLE 3 Unit 1 Unit 2 Unit 3 Unit 4 pH Probes: Hamilton Easy Ferm PlusK8 200 S/N 18533 18380 18560 18562 Offset 0.061 −0.029 0.038 0.02 Slope99.10% 97.55% 97.55% 97.97% RD Probes: Mettler ToledoPt4805-SC-DPAS-K8S/225 S/N 5501480 5482413 5482414 N/A Offset 5.0487.665 8.803 N/A

Headplate/Tubing: The vessel headplate and tubing was prepared, cleanedand inspected prior to the run. All luer valves were replaced from theprevious run.

Autoclaving/Setup: The vessels were autoclaved per the setup procedure,each containing 300 mL of 1×PBS which was decanted prior to filling withmedia. Bottles for base were prepared, 1 bottle for each vessel. Bottleswere filled in the biosafety cabinet with 10N sodium hydroxide, however,the sodium hydroxide was not sterile filtered due to the viscosity. Alllines were connected, and control loops initialized prior toinoculation. All process parameters were allowed to reach equilibriumprior to inoculation.

Inoculum: The inoculating culture (Blautia massiliensis strain A) forD002 was prepared, in BM1 media, and grew for 12 hours. Then, 10 mL ofthe culture was added to each vessel. The OD of the inoculating culturewas 0.658. Result of inoculum growth were recorded in the strain bankmaster file. The pH of all vessels was approx. 7.00 at the time ofinoculation.

Process Parameters: The following process parameters were used duringthe run.

Parameters were not changed after inoculation.

Temperature: 37C

Sparger Flow: 145 mL/minOverlay Flow: 85 mL/min

Agitation: 150 RPM

pH:

Vessel 1: 5.20 Vessel 2: 5.80 Vessel 3: 6.40 Vessel 4: 7.00

The details about the process are below.

pH control at 7.0

Temperature 37 C

Inoculum size 1%

Gas 90% N2+5% CO2 Sparging 0.1 vvm Agitation 300 rpm

Sampling and Analysis: Sampling and Analysis was performed. Anadditional five vacutainer samples were taken from each vessel duringsamples 1 and 3 to be used for downstream analysis. Ten additionalvacutainer samples were taken from each vessel during sample 2 for thesame purpose. A vacutainer sample was taken from each vessel immediatelyprior to takedown for quality control analysis via 16S PCR. Samplingtimes are listed in Table 4 below.

TABLE 4 Sampling Run Time Time (DD:HH:MM) Sample Vessel PVs OK? Notes 8:30 00:11:47 1 1, 2, 3, 4 OK 12:41 00:15:15 2 1, 2, 3, 4 OK 15:3600:18:11 3 1, 2, 3, 4 OK

Reactor Takedown: The run was stopped a day later and all takedownprocedures performed.

Critical Process Parameters: D002 passes all critical process parameterchecks.

Data: A selection of data is reproduced in its un-manipulated form belowfor ease of reference.

TABLE 5 CFU CFU av RSD Sample Vessel (cell/mL) SD (%) Note* 1 1 2.97E+098.02E+08 27.03642565 2 4.10E+09 5.29E+08 12.90610396 3 4.90E+09 4.58E+089.352195296 4 2.60E+09 4.58E+08 17.62529113 2 1 2.33E+08 1.15E+0849.48716593 2 7.33E+08 3.51E+08 47.88933524 3 4.33E+08 5.77E+0713.32346775 4 9.67E+08 3.51E+08 36.32984053 3 1 1.30E+09 5.20E+0839.97040325 2 3.60E+09 4.00E+08 11.11111111 3 1.90E+09 2.65E+0813.9250069 4 2.53E+09 4.04E+08 15.95309954

TABLE 6 Optical Density Sample Vessel Dilution Average Stdev Note* 1 120x 5.18 0.03 2 20x 5.98 0.07 3 20x 5.57 0.11 4 20x 5.51 0.01 2 1 20x6.96 0.05 2 20x 5.59 0.06 3 20x 6.62 0.05 4 20x 6.97 0.17 3 1 20x 6.370.09 2 20x 5.36 0.03 3 20x 6.44 0.14 4 20x 6.81 0.14

Results:

FIG. 9 shows anaerobic bioreactor process at controlled pH at differentset points. FIG. 10 shows total base addition during fermentationprocesses; samples S1 and S3 were taken at designated points. FIG. 11shows cell recovery after downstream processing. FIG. 12 shows relativecell viability of samples 51 and S3 after downstream processing.

Conclusions: pH data indicates that the used PID settings providedrobust control of pH in the DasGip system (FIG. 9). Total base additionin FIG. 10 may show a metabolic switch occurring as cells switch fromexponential to stationary phase. bacteria grew in bioreactors up to5×10⁹ CFU per mL (FIG. 11). The growth was similar at different pH setpoints, however, the recovery after air ambient downstream processingwas significantly influenced by bioreactor pH (FIG. 11). An early pointof harvest resulted better recovery (FIG. 12).

Example 4: Orally Administered Blautia Strain A Inhibits ColorectalCarcinoma Tumor Growth

Female 6-8 week old Balb/c mice were obtained from Taconic (Germantown,N.Y.). 100,000 CT-26 colorectal tumor cells (ATCC CRL-2638) wereresuspended in sterile PBS and inoculated in the presence of 50%Matrigel. CT-26 tumor cells were subcutaneously injected into one hindflank of each mouse. When tumor volumes reached an average of 100 mm³(approximately 10-12 days following tumor cell inoculation), animalswere distributed into the following groups: 1) Vehicle+PBS; 2) anti-PD-1antibody; and 3) Blautia massiliensis Strain A (ATCC Deposit NumberPTA-125134). Antibodies were administered intraperitoneally (i.p.) at100 ug/mouse (100 ul final volume) every four days, starting on day 1,and Blautia Strain A bacteria (5.5×10⁴) were administered by oral gavage(p.o.) daily, starting on day 1 until the conclusion of the study. TheBlautia Strain A group showed tumor growth inhibition comparable to thatseen in the anti-PD-1 group (FIGS. 1 and 2).

Example 5: Blautia Strain A in a Mouse Melanoma Model

Female 6-8 week old C57Bl/6 mice were obtained from Taconic (Germantown,N.Y.). 100,000 B16-F10 (ATCC CRL-6475) tumor cells were resuspended insterile PBS containing 50% Matrigel and inoculated in a 100 ul finalvolume into one hind flank (the first flank) of each mouse. Animals weredistributed into the following groups: 1) Vehicle+PBS; 2) anti-PD-L1antibody; and 3) Blautia massiliensis Strain A (ATCC Deposit NumberPTA-125134). Antibodies were administered intraperitoneally (i.p.) at100 ug/mouse (100 ul final volume) every four days, starting on day 1,and Blautia Strain A bacteria (6.1×10⁷) were administered by oral gavage(p.o.) daily, starting on day 1 until the conclusion of the study. TheBlautia Strain A group showed better tumor growth inhibition compared tothat seen in the anti-PD-L1 group (FIGS. 3 and 4).

Example 6: Treatment of Melanoma Using Blautia massiliensis Strain A inCombination with Anti-PD-L1

As described in Example 5 B16-F10 (ATCC CRL-6475) tumor cells wereinoculated into one hind flank (the first flank) of mice and animalswere distributed into the following groups: 1) Vehicle+PBS; 2)vehicle+anti-PD-L1 antibody; 3) Blautia massiliensis Strain A+PBS; and4) Blautia massiliensis Strain A+anti-PD-L1. Antibodies wereadministered intraperitoneally (i.p.) at 100 ug/mouse (100 ul finalvolume) every four days, starting on day 1, and Blautia Strain Abacteria (6.1×10⁷) were administered by oral gavage (p.o.) daily,starting on day 1 until the conclusion of the study. The Blautia StrainA+anti-PD-L1 group showed the best tumor growth inhibition (FIG. 5).

Example 7: Infiltration of CD3+ Cells is Significantly Increased in CT26and B16-F10 Tumors in Mice Orally Gavaged with Blautia massiliensisStrain A

In some studies, at various timepoints, mice are sacrificed and tumors,lymph nodes, or other tissues may be removed for ex vivo flow cytometricanalysis using methods known in the art. For example, tumors aredissociated using a Miltenyi tumor dissociation enzyme cocktailaccording to the manufacturer's instructions. Tumor weights are recordedand tumors are chopped then placed in 15 ml tubes containing the enzymecocktail and placed on ice. Samples are then placed on a gentle shakerat 37° C. for 45 minutes and quenched with up to 15 ml complete RPMI.Each cell suspension is strained through a 70 μm filter into a 50 mlfalcon tube and centrifuged at 1000 rpm for 10 minutes. Cells areresuspended in FACS buffer and washed to remove remaining debris. Ifnecessary, samples are strained again through a second 70 μm filter intoa new tube. Cells are stained for analysis by flow cytometry usingtechniques known in the art. Staining antibodies can include anti-CD11c(dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII,anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzedinclude pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8,CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), andmacrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1,Gr-1). In addition to immunophenotyping, cytokines are analyzedincluding, but not limited to, IP-10, TNFa, IL-17, IL-13, IL-12p70,IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF,M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1, using techniques known inthe art. Cytokine analysis may be analyzed on immune cells obtained fromlymph nodes or other tissue, tumor sections, dissociated tumor cells,and/or on purified CD45+ tumor-infiltrated immune cells obtained exvivo. Cytokine levels may be ascertained using various stainingtechniques, qPCR, or other techniques known in the arts.Immunohistochemistry may also be performed on tumor sections to measureT cells, macrophages, dendritic cells, and checkpoint molecule or otherprotein expression using techniques known in the art.

Mice were inoculated with CT26 or B16-F10 tumor cells, divided intogroups as described above, and orally gavaged with Blautia massiliensisStrain A 8.3×10{circumflex over ( )}9 CFU/mouse daily. Other mice weregiven vehicle as a negative control. Mice from this study weresacrificed on day 10 and tumors harvested for analysis. Using knowntechniques described above, tumor sections were stained with anti-CD3antibodies (T cell marker) to assess the average CD3+ cells per tumorsection, and tumor gene expression was analyzed by qPCR using a TaqManassay as follows: Tumor RNA was isolated with Qiagen's RNeasy Mini kit(Catalog No. 74104), using the manufacturer's protocol. cDNA wasisolated using BioRad's iScript™ cDNA Synthesis Kit according to themanufacturer's protocol (Catalog No. 1708891). The qPCR reaction was runusing SsoAdvanced™ Universal Probes Supermix 1725281 usingmanufacturer's protocol on the BioRad CFX384 instrument. The TaqManmouse specific probes were from Invitrogen, MHC I (b2m) Mm00437762_m1and β-actin Mm01205647_g1 for normalizing gene expression. The delta Ctvalue for each tumor was calculated for MHC I using β-actin as thereference gene. The expression for MHC I was calculated using theformula 2{circumflex over ( )}-delta Ct for each mouse tumor and thisvalue was divided by its respective final tumor volume at the end of thestudy.

Tumor volumes were taken prior to CD3+ immune cell infiltrate and MHCClass I expression analysis. Infiltration of CD3+ immune cells wassignificantly increased in the Blautia massiliensis Strain A grouprelative to vehicle in a mouse colorectal carcinoma model (FIG. 6) andin mouse melanoma model (FIG. 7). Blautia massiliensis Strain A induceda striking upregulation of MHC Class I expression (FIG. 8).

Rather than being sacrificed, some mice may be rechallenged with tumorcell injection into the contralateral flank (or other area) to determinethe impact of the immune system's memory response on tumor growth.

Example 8: Blautia Strain A in a Mouse Lung Cancer Model

Blautia Strain A is tested for its efficacy in the mouse lung cancermodel, either alone or in combination with other cancer therapies,including checkpoint inhibitor(s). Mice are divided into groupsreceiving Blautia Strain A, with or without checkpoint inhibitortreatment. Blautia Strain A is administered at varied doses at definedintervals. For example, some mice receive Blautia Strain A (p.o.) on theday following tumor cell injection (day 1). Some mice receive seven (7)consecutive doses of Blautia Strain A (one dose per day on days 14-21).Other mice receive daily dosing or, alternatively, some mice receivedosing every other day. Alternatively, mice are randomized into varioustreatment groups at a defined timepoint (e.g. on day 13) or when thetumors reach a certain size (e.g. 100 mm³) and treatment is theninitiated accordingly.

1×10⁶ LLC1 cells, or an appropriate number of lung cancer cells fromanother lung cancer cell line, are injected into the hind flank ofsyngeneic mice. Tumors from the various treatment groups are measuredwith calipers at regular intervals. Some mice are sacrificed for ex vivotumor analysis using flow cytometry. Other mice may be rechallenged withtumor cell injection into the contralateral flank to determine theimpact of the immune system's memory response on tumor growth.

Example 9: Blautia Strain A in a Mouse Breast Cancer Model

Blautia Strain A is tested for its efficacy in the mouse breast cancermodel, either alone or in combination with other cancer therapies,including checkpoint inhibitor(s). Mice are divided into groupsreceiving Blautia Strain A, with or without checkpoint inhibitortreatment. Blautia Strain A is administered at varied doses at definedintervals. For example, some mice receive Blautia Strain A (p.o.) on theday following tumor cell injection (day 1). Some mice receive seven (7)consecutive doses of Blautia Strain A (one dose per day on days 14-21).Other mice receive daily dosing or, alternatively, some mice receivedosing every other day. Alternatively, mice are randomized into varioustreatment groups at a defined timepoint (e.g. on day 13) or when thetumors reach a certain size (e.g. 100 mm³) and treatment is theninitiated accordingly.

4T1 mouse mammary carcinoma cells are obtained from ATCC and 1×10⁶ cellsin 50 ul PBS are injected subcutaneously into one or both hind limbs ofBalb/c female mice (as described by Wang et al. 2003, Systemicdissemination of viral vectors during intratumoral injection. MolecularCancer Therapeutics; 2(11)). Alternatively, EMT6 mouse mammary carcinomacells are obtained from ATCC and 1×10⁶ cells in 50 μl PBS are injectedsubcutaneously into one or both of the hind limbs of Balb/c female mice6-8 weeks old (as described by Guo et al. 2014, CombinatorialPhotothermal and Immuno Cancer Therapy Using Chitosan-Coated HollowCopper Sulfide Nanoparticles. ASC Nano.; 8(6): 5670-5681). In addition,other available mouse mammary cell lines may be used.

Tumors from the various treatment groups are measured with calipers atregular intervals. Blautia Strain A is administered at varied doses atdefined intervals. For example, some mice are sacrificed for ex vivotumor analysis using flow cytometry. Other mice may be rechallenged withtumor cell injection into the contralateral flank to determine theimpact of the immune system's memory response on tumor growth.

Alternatively, 4T1 cells can be used in an orthotopic murine model ofbreast cancer as described by Tao et al. (Tao et al. 2008. Imagable 4T1model for the study of late stage breast cancer. 8: 288). Mice aresacrificed for ex vivo tumor analysis. Tumors are analyzed by flowcytometry and immunohistochemistry.

Example 10: Blautia Strain A in a Mouse Pancreatic Cancer Model

Blautia Strain A is tested for its efficacy in the mouse model ofpancreatic cancer, either alone or in combination with other cancertherapies, including checkpoint inhibitor(s). Mice are divided intogroups receiving Blautia Strain A, with or without checkpoint inhibitortreatment. Some mice receive Blautia Strain A (p.o.) on the dayfollowing tumor cell injection (day 1). Some mice receive seven (7)consecutive doses of Blautia Strain A (one dose per day on days 14-21).Other mice receive daily dosing or, alternatively, some mice receivedosing every other day. Alternatively, mice are randomized into varioustreatment groups at a defined timepoint (e.g. on day 13) or when thetumors reach a certain size (e.g. 100 mm³) and treatment is theninitiated accordingly.

Panc02 cells are maintained in DMEM, supplemented with 10% fetal calfserum and 1% penicillin/streptomycin, and incubated at 37° C. at 5% CO2.Female 8-10 week-old C57Bl/6 mice are obtained from Charles River, Inc.or other certified vendor. Female C57Bl/6 mice are injectedsubcutaneously into the right hind flank with 1×10⁶Panc02 cells. Thisprotocol is based on standard Panc02 tumor models (Maletzki et al. 2008.Pancreatic cancer regression by intratumoral injection of livestreptococcus pyogenes in a syngeneic mouse model. Gut. 57:483-491).Tumors from the various treatment groups are measured with calipers atregular intervals. Some mice are sacrificed for ex vivo tumor analysisusing flow cytometry, while other mice are rechallenged to determine theimpact of the memory response on tumor growth.

Alternatively, Panc02, 6606PDA, or Capan-1 cells lines can be used in anorthotopic murine model of pancreatic cancer as described by Partecke etal. (Partecke et al. 2011. A syngeneic orthotopic murine model ofpancreatic adenocarcinoma in the C57/B16 mouse using the Panc02 and6606PDA cell lines. Eur. Surg. Res. 47(2):98-107) or Chai et al. (Chaiet al. 2013. Bioluminescent orthotopic model of pancreatic cancerprogression. J. Vis. Exp. 76: 50395). Mice are sacrificed for ex vivotumor analysis. Tumors are analyzed by flow cytometry andimmunohistochemistry.

Example 11: Blautia Strain A in a Mouse Model of HepatocellularCarcinoma

Blautia Strain A is tested for its efficacy in the mouse model ofhepatocellular carcinoma, either alone or in combination with othercancer therapies, including checkpoint inhibitor(s). Mice are dividedinto groups receiving Blautia Strain A, with or without checkpointinhibitor treatment. Blautia Strain A is administered at varied doses atdefined intervals. For example, some mice receive Blautia Strain A(p.o.) on the day following tumor cell injection (day 1). Some micereceive seven (7) consecutive doses of Blautia Strain A (one dose perday on days 14-21). Other mice receive daily dosing or, alternatively,some mice receive dosing every other day. Alternatively, mice arerandomized into various treatment groups at a defined timepoint (e.g. onday 13) or when the tumors reach a certain size (e.g. 100 mm³) andtreatment is then initiated accordingly.

Hepatocellular carcinoma is induced in mice by subcutaneous inoculationof 1×10⁶Hepa129 cells (obtained from NCI or other source), or anappropriate number of cells from other hepatocellular carcinoma cellline (as described by Gonzalez-Carmona et al. 2008. CD40ligand-expressing dendritic cells induce regression of hepatocellularcarcinoma by activating innate and acquired immunity in vivo.Hepatology. 48(1):157-168). Tumor cells are inoculated into one or bothflanks. Tumors from the various treatment groups are measured withcalipers at regular intervals. Some mice are sacrificed for ex vivotumor analysis using flow cytometry, while other mice are rechallengedto determine the impact of the memory response on tumor growth.

Example 12: Blautia Strain A in a Mouse Lymphoma Model

Blautia Strain A is tested for its efficacy in the mouse model oflymphoma, either alone or in combination with other cancer therapies,including checkpoint inhibitor(s). Mice are divided into groupsreceiving Blautia Strain A, with or without checkpoint inhibitortreatment. Blautia Strain A is administered at varied doses at definedintervals. For example, some mice receive Blautia Strain A (p.o.) on theday following tumor cell injection (day 1). Some mice receive seven (7)consecutive doses of Blautia Strain A (one dose per day on days 14-21).Other mice receive daily dosing or, alternatively, some mice receivedosing every other day. Alternatively, mice are randomized into varioustreatment groups at a defined timepoint (e.g. on day 13) or when thetumors reach a certain size (e.g. 100 mm³) and treatment is theninitiated accordingly.

One lymphoma cell line is the A20 lymphoma, although other lymphoma celllines may be used with syngeneic mice. A20 lymphoma cells are obtainedfrom ATCC and 5×10⁶ cells in 50 ul PBS are injected subcutaneously intoone or both of the hind limbs of Balb/c female mice (as described byHouot et al. 2009. T-cell modulation combined with intratumoral CpGcures lymphoma in a mouse model without the need for chemotherapy.Blood. 113(15): 3546-3552). Tumors from the various treatment groups aremeasured with calipers at regular intervals. Some mice are sacrificedfor ex vivo tumor analysis using flow cytometry, while other mice arerechallenged to determine the impact of the memory response on tumorgrowth.

Example 13: Blautia Strain A in a Mouse Prostate Cancer Model

Blautia Strain A is tested for its efficacy in the mouse model ofprostate cancer, either alone or in combination with other cancertherapies, including checkpoint inhibitor(s). Mice are divided intogroups receiving Blautia Strain A, with or without checkpoint inhibitortreatment. Blautia Strain A is administered at varied doses at definedintervals. For example, some mice receive Blautia Strain A (p.o.) on theday following tumor cell injection (day 1). Some mice receive seven (7)consecutive doses of Blautia Strain A (one dose per day on days 14-21).Other mice receive daily dosing or, alternatively, some mice receivedosing every other day. Alternatively, mice are randomized into varioustreatment groups at a defined timepoint (e.g. on day 13) or when thetumors reach a certain size (e.g. 100 mm³) and treatment is theninitiated accordingly.

Mouse prostate cancer cells (1×10⁵ RM-1 cells or an appropriate numberof cells from another prostate cancer cell line) are injected intosyngeneic mice. Tumors from the various treatment groups are measuredwith calipers at regular intervals. Some mice are sacrificed for ex vivotumor analysis using flow cytometry, while other mice are rechallengedto determine the impact of the memory response on tumor growth.

Example 14: Blautia Strain A in a Mouse Plasmacytoma Model

Blautia Strain A is tested for its efficacy in the mouse model ofplasmacytoma, either alone or in combination with other cancertherapies, including checkpoint inhibitor(s). Mice are divided intogroups receiving Blautia Strain A, with or without checkpoint inhibitortreatment. Blautia Strain A is administered at varied doses at definedintervals. For example, some mice receive Blautia Strain A (p.o.) on theday following tumor cell injection (day 1). Some mice receive seven (7)consecutive doses of Blautia Strain A (one dose per day on days 14-21).Other mice receive daily dosing or, alternatively, some mice receivedosing every other day. Alternatively, mice are randomized into varioustreatment groups at a defined timepoint (e.g. on day 13) or when thetumors reach a certain size (e.g. 100 mm³) and treatment is theninitiated accordingly.

Mineral Oil Induced Model of Plasmacytoma

To examine the efficacy of Blautia Strain A in a plasmacytoma ormultiple myeloma model, mice are injected intraperitoneally three timeswith 500 ul of 2,6,10,12-tetramethylpentadecane (“pristane oil”) atvarious time points between 0 and 60 days, as described by Potter et al.1983. Peritoneal plasmacytomagenesis in mice: comparison of differentpristane dose regimens. J. Natl. Cancer Inst. 71(2):391-5 (see alsoLattanzio et al. 1997. Defective Development of Pristane-Oil InducedPlasmacytomas in Interleukin-6-Deficient BALB/C Mice. Am. J. Pathology:151(3):689696). Progression of disease is measured by the degree ofabdominal swelling and immune cells and particles in the ascites.Ascites fluid is analyzed for immune cell phenotype by flow cytometry.

Cell-Line Induced Model of Plasmacytoma

To examine the efficacy of Blautia Strain A in a plasmacytoma ormultiple myeloma model, either MOPC-104E cells or J558 plasmacytomacells (TIB-6 ATCC) are injected subcutaneously into one or more hindflanks of Balb/c mice (5×10⁶ cells), based on model described byBhoopalam et al. 1980. Effect of dextran-S(alpha, 1-3 dextran) on thegrowth of plasmacytomas MOPC-104E and J558. J. Immunol. 125(4):1454-8(see also Wang et al. 2015. IL-10 enhances CTL-mediated tumor rejectionby inhibiting highly suppressive CD4+ T cells and promoting CTLpersistence in a murine model of plasmacytoma. OncoImmunology. 4(7):e1014232-1-9). Mice are divided into groups receiving Blautia Strain Aby oral gavage, and with or without checkpoint inhibitor treatment.Tumors from the various treatment groups are measured with calipers atregular intervals. Some mice are sacrificed for ex vivo tumor analysisusing flow cytometry, while other mice are rechallenged to determine theimpact of the memory response on tumor growth.

Example 15: Blautia Strain A in a SCID Mouse Model of Mouse Myeloma

Blautia Strain A is tested for its efficacy in the SCID mouse model ofmyeloma, either alone or in combination with other cancer therapies,including checkpoint inhibitor(s). Mice are divided into groupsreceiving Blautia Strain A, with or without checkpoint inhibitortreatment. Blautia Strain A is administered at varied doses at definedintervals. For example, some mice receive Blautia Strain A (p.o.) on theday following tumor cell injection (day 1). Some mice receive seven (7)consecutive doses of Blautia Strain A (one dose per day on days 14-21).Other mice receive daily dosing or, alternatively, some mice receivedosing every other day. Alternatively, mice are randomized into varioustreatment groups at a defined timepoint (e.g. on day 13) or when thetumors reach a certain size (e.g. 100 mm³) and treatment is theninitiated accordingly.

To examine the efficacy of Blautia Strain A using a human plasma cellleukemia, 1×10⁷ human myeloma cell lines, ARH77 cells (ARH77-ATCCCRL-1621, or an appropriate number of cells from another myeloma cellline such as KPMM2) are used. Myeloma cells are injected subcutaneouslyinto one or both hind flanks of SCID mice (See Caers et al. 2004. Ofmice and men: disease models of multiple myeloma. Drug Discovery Today:Disease Models. 1(4):373-380. Tumors from the various treatment groupsare measured with calipers at regular intervals. Some mice aresacrificed for ex vivo tumor analysis using flow cytometry, while othermice are rechallenged to determine the impact of the memory response ontumor growth.

Example 16: Blautia Strain A in a Mouse Renal Cell Carcinoma Model

Blautia Strain A is tested for its efficacy in the mouse model of renalcell carcinoma, either alone or in combination with other cancertherapies, including checkpoint inhibitor(s). Mice are divided intogroups receiving Blautia Strain A, with or without checkpoint inhibitortreatment. Blautia Strain A is administered at varied doses at definedintervals. For example, some mice receive Blautia Strain A (p.o.) on theday following tumor cell injection (day 1). Some mice receive seven (7)consecutive doses of Blautia Strain A (one dose per day on days 14-21).Other mice receive daily dosing or, alternatively, some mice receivedosing every other day. Alternatively, mice are randomized into varioustreatment groups at a defined timepoint (e.g. on day 13) or when thetumors reach a certain size (e.g. 100 mm³) and treatment is theninitiated accordingly.

To examine the efficacy of Blautia Strain A in a mouse model of renalcell carcinoma, Renca cells (ATCC CRL-2947) or other renal cellcarcinoma cells are injected subcutaneously into one or both flanks of7-8 week old syngeneic Balb/c mice (5×10⁶ in 0.1 ml PBS). Tumors fromthe various treatment groups are measured with calipers at regularintervals. Some mice are sacrificed for ex vivo tumor analysis usingflow cytometry, while other mice are rechallenged to determine theimpact of the memory response on tumor growth.

Example 17: Blautia Strain A in a Mouse Bladder Cancer Model

Blautia Strain A is tested for its efficacy in the mouse model ofbladder cancer, either alone or in combination with other cancertherapies, including checkpoint inhibitor(s). Mice are divided intogroups receiving Blautia Strain A, with or without checkpoint inhibitortreatment. Blautia Strain A is administered at varied doses at definedintervals. For example, some mice receive Blautia Strain A (p.o.) on theday following tumor cell injection (day 1). Some mice receive seven (7)consecutive doses of Blautia Strain A (one dose per day on days 14-21).Other mice receive daily dosing or, alternatively, some mice receivedosing every other day. Alternatively, mice are randomized into varioustreatment groups at a defined timepoint (e.g. on day 13) or when thetumors reach a certain size (e.g. 100 mm³) and treatment is theninitiated accordingly.

On the day of inoculation, MBT-2 cells (or other bladder cancer cellline) are harvested and resuspended in 1:1 PBS/Matrigel mixture.2×10⁵MBT-2 cells are suspended in 100 ul of mixture and injectedsubcutaneously into one or both hind flanks of syngeneic mice. Tumorsare measured with calipers at regular intervals.

Some mice are sacrificed for ex vivo tumor analysis using flowcytometry, while other mice are rechallenged to determine the impact ofthe memory response on tumor growth.

Example 18: The Efficacy of a Blautia Strain A Strain is Compared toOther Blautia Strains for Efficacy in Cancer Models

Using methods described above, various Blautia Strain A and otherBlautia strains are tested and compared for efficacy in various cancermodels. Such models may include, but are not limited to, melanoma, lungcancer, breast cancer, colon cancer, colorectal cancer, pancreaticcancer, hepatocellular cancer, lymphoma, prostate cancer, plasmacytoma,a SCID model of myeloma, renal cell carcinoma, and/or bladder cancer.

Example 19: An Open-Label Study of the Safety, Tolerability and Efficacyof a Blautia Strain A Oral Therapeutic in Patients with MetastaticColorectal Carcinoma

A multi-center, open-label clinical study with dose escalations and doseexpansions to assess preliminary safety, tolerability, and efficacy ofthe Blautia Strain A is performed. The study proceeds in three parts:Part A: Dose escalation in microsatellite stable (MSS) metastaticcolorectal carcinoma (CRC) and alternative indications, Part B: MSS CRCand alternative indications, and Part C: microsatellite instable (MSI)CRC. Dose escalation occurs in a standard 3+3 design, as set forth inTable 4. In part A, from 9 to 18 patients are enrolled. Parts B and Care enrolled concurrently. In Part B, up to 15 and no fewer than 10patients are enrolled. In Part C no fewer than 5 and up to 10 patientsare enrolled. Based on in vivo studies of allograft bearing mice, anexemplary estimated human effective dose is 2×10¹¹ organisms per day,delivered per enteric capsule.

The primary objectives of the study include the determination of themaximum tolerated dose within the tested dose range for Blautia Strain Ain patients with advanced malignancies; the determination of therecommended phase 2 dose for Blautia Strain A in CRC; the evaluation ofthe safety and tolerability, including dose limiting toxicities ofBlautia Strain A; and the demonstration of anti-tumor activity ofBlautia Strain A in patients with CRC (MSI and MSS). The anti-tumoractivity will be assessed by monitoring changes in circulating tumorcells, immune cell subsets in blood and tumor, and tumor viability;objective response rate and duration of response; progression-freesurvival; overall survival; clinical benefit rate and duration ofclinical benefit rate; and disease control rate and duration of diseasecontrol rate. The secondary objective is to determine the humandistribution and elimination of Blautia Strain A. The exploratoryobjectives is to evaluate correlations of Blautia Strain A response withmolecular markers relevant to each tumor type and to identify possiblemechanisms of resistance to Blautia Strain A.

Inclusion and Exclusion Criteria:

The inclusion criteria for all parts of the study include the following:

-   -   1. Written informed consent obtained prior to any screening        procedures and in accordance with federal, local, and        institutional guidelines.    -   2. Age ≥18 years.    -   3. Adequate hepatic function:        -   a. total bilirubin ≤2 times the upper limit of normal (ULN)            (except patients with Gilbert's syndrome [hereditary            indirect hyperbilirubinemia] who must have a total bilirubin            of ≤3 times ULN),        -   b. aspartate aminotransferase (AST) and alanine            aminotransferase (ALT)≤2.5 times ULN (except patients with            known liver involvement of their tumor who must have their            AST and ALT ≤5.0 times ULN).    -   4. Adequate renal function: estimated creatinine clearance of        ≥30 mL/min, calculated using the formula of Cockcroft and Gault        (140−Age)·Mass (kg)/(72·creatinine mg/dL); multiply by 0.85 if        female.    -   5. All patients in part B+C must be willing to have fresh        biopsies at start of therapy and after 4 weeks of therapy.    -   6. Contraception: Female patients of child-bearing potential        must agree to use dual methods of contraception (including one        highly effective and one effective method of contraception) and        have a negative serum pregnancy test at Screening, and male        patients must use an effective barrier method of contraception        if sexually active with a female of child-bearing potential. For        both male and female patients, effective methods of        contraception must be used throughout the study and for 3 months        following the last dose.

For the portions of the study testing relapsed/refactory colorectalcancer, the inclusion criteria also include the following:

-   -   7. Histological or cytological documentation of adenocarcinoma        of the colon or rectum.    -   8. Known MSI/MSS status.    -   9. Measurable disease by RECIST v1.1.    -   10. Metastatic disease not suitable for upfront curative-intent        surgery.    -   11. Documented evidence of progressive disease according to        RECIST v1.1.    -   12. Prior treatment (with completion of a course of therapy, or        to disease progression or intolerability) with each of the        following:        -   a. Fluoropyrimidine-, oxaliplatin-, irinotecan-based            chemotherapies (e.g., FOLFOX and FOLFIRI)        -   b. if KRAS wild-type, an anti-EGFR therapy,        -   c. Regorafenib or TAS 102 (Past A required, parts B+C            optional)        -   d. Radiation and surgery are not considered as prior            anticancer regimens    -   13. Patients should not be transfusion dependent.    -   14. Adequate hematopoietic function: ANC≥1000/mm3, hemoglobin        (Hb)≥9.0 g/dL, and platelet count ≥100,000/mm3.    -   15. Eastern Cooperative Oncology Group (ECOG) performance status        of ≤1.    -   16. Life expectancy of ≥3 months.

The following categories of patient are excluded from the study:

-   -   1. Female patients who are pregnant or lactating.    -   2. Major surgery within 4 weeks before C1D1.    -   3. Impaired cardiac function or clinically significant cardiac        diseases, including any of the following:        -   a. Unstable angina or acute myocardial infarction ≤3 months            prior to C1D1;        -   b. Clinically significant heart disease (e.g., symptomatic            congestive heart failure [e.g., >NYHA Class 2]; uncontrolled            arrhythmia, or hypertension; history of labile hypertension            or poor compliance with an antihypertensive regimen).    -   4. Uncontrolled active severe systemic infection requiring        parenteral antibiotics, antivirals, or antifungals within one        week prior to C1D1.    -   5. Any ongoing antibiotic treatment which has not been        discontinued at least 3 days prior to initiation of therapy    -   6. Patients with known symptomatic brain metastasis are not        suitable for enrollment. Patients with asymptomatic, stable,        treated brain metastases are eligible for study entry.    -   7. Patients with a known history of human immunodeficiency virus        (HIV); HIV testing is not required as part of this study.    -   8. Known, active hepatitis A, B, or C infection; or known to be        positive for HCV RNA or HBsAg (HBV surface antigen).    -   9. Prior malignancies:        -   a. Patients with adequately resected basal or squamous cell            carcinoma of the skin, or adequately resected carcinoma in            situ (i.e. cervix) may enroll irrespective of the time of            diagnosis.        -   b. Prior malignancies which may interfere with the            interpretation of the study. Cancer treated with curative            intent <5 years previously will not be allowed unless            approved by the Sponsor. Cancer treated with curative            intent >5 years previously and without evidence of            recurrence will be allowed.    -   10. Patients with active central nervous system (CNS)        malignancy. Patients who have only had prophylactic intrathecal        or intravenous chemotherapy against CNS disease are eligible.    -   11. Patients with gastrointestinal tract disease (or        uncontrolled vomiting or diarrhea) that could interfere with the        absorption of EVP001.    -   12. Serious psychiatric or medical conditions that, in the        opinion of the Investigator, could interfere with treatment,        compliance, or the ability to give consent.    -   13. Patients unwilling to comply with the protocol including        required biopsies and sample collections required to measure        disease.    -   14. Radiotherapy within two weeks prior to screening. Patients        must have recovered from clinically significant toxicities.

The use of any concomitant medication/therapy, includingover-the-counter (OTC) medications deemed necessary for the care of thepatient is permitted during the study. Medications required to treatAEs, manage cancer symptoms, concurrent stable diseases and supportivecare agents (e.g. blood product transfusions), pain medications,anti-emetics, and anti-diarrheals are allowed. Concurrent therapy withgrowth factors is allowed. The use of any immunosuppressive agents mustbe discussed between the Investigator and the Medical Monitor on acase-by-case basis. Any diagnostic, therapeutic, or surgical procedureperformed during the study period should be recorded, including thedates, description of the procedure(s), and any clinical findings, ifapplicable. All antibiotics are contraindicated.

Hormonal contraceptives are permitted in women of child-bearingpotential. Hormonal contraceptives include any marketed contraceptiveagent that includes an estrogen and/or a progestational agent.

Investigational or commercial anticancer agents other than BlautiaStrain A is not allowed during the study. The initiation of anynon-protocol specific anti-tumor treatment is considered an indicationof disease relapse/progression and should be recorded appropriately inthe electronic case report forms.

Palliative radiation therapy to non-target lesions is permitted butstudy treatment is held for ≥1 day before the start of palliativeradiation therapy and ≥1 day following each dose of palliative radiationtherapy. Treatment with Blautia Strain A is not discontinued solely dueto palliative radiation.

Supportive measures for optimal medical care should be provided topatients during participation in this study. These should be based oninstitutional and/or National Comprehensive Cancer Network (NCCN)guidelines.

Dose Escalation Study

Patients receive all Blautia Strain A doses during the 7 day treatmentperiod, or have had a dose-limiting toxicity (DLT) within the treatmentperiod to be considered evaluable for dose escalation decisions. BlautiaStrain A is orally administered as tablets or enteric coated capsules.Dose escalation decisions occur when the cohort of patients has metthese criteria.

A DLT is defined as an adverse event (AE) or abnormal laboratory valuethat occurs within the first 7 days of treatment with Blautia Strain A,except for those that are clearly and incontrovertibly due to underlyingdisease, disease progression, or extraneous causes, and meets any of thecriteria included in Table 7. National Cancer Institute CommonTerminology Criteria for Adverse Events (NCI CTCAE) (Version 4.03) isused for all grading. In addition, >4 missed doses of Blautia Strain Awill be considered a DLT. Dose escalation decisions occur when thecohort of patients has met these criteria.

TABLE 7 Criteria for defining DLT Any of the following criteria (basedon CTCAE [Version Toxicity 4.03]): Non- Grade ≥ 3 nausea/vomiting, whiletaking optimal Hematologic supportive medications. Any other Grade ≥ 3non-hematological toxicity except alopecia or electrolyte abnormalitiescorrectable with supportive therapy. Hematologic Grade 4 neutropenialasting more than 5 days. Febrile neutropenia (absolute neutrophil count[ANC] < 1 × 10⁹/L, fever > 38.5° C.). Grade 4 thrombocytopenia, or Grade3 thrombocytopenia with bleeding, or any requirement for platelettransfusion. CTCAE Version 4.03 will be used for grading all AEs andlaboratory abnormalities. Patients may receive supportive care as perlocal institutional guidelines.

To implement dose escalation decisions, the available toxicityinformation (i.e., all AEs and all laboratory abnormalities regardlessof DLT assessment) is evaluated by the enrolling Investigators andSponsor medical monitor at a dose decision meeting or teleconference.Decisions are based on an evaluation of all relevant data available fromall dose cohorts evaluated in the ongoing study including safetyinformation, DLTs, all NCI CTCAE, Version 4.03 toxicity data duringCycle 1 from evaluable patients. Drug administration at the next higherdose cohort may not proceed until the Investigator receives writtenconfirmation from Sponsor indicating that the results of the previousdose cohort were evaluated and that it is permissible to proceed to thenext higher dose cohort.

Table 8 below describes the starting dose and the dose levels that maybe evaluated during the study for all parts of the study.

TABLE 8 Blautia Strain A Dose Escalation Levels Dose Levels Cohort QDdosing; 3 week cycles Number of Patients 1 2 × 10⁸ organisms 3 + 3 2 1 ×10⁹ organisms 3 + 3 3 2 × 10⁹ organisms 3 + 3 4 (optional) 4 × 10⁹organisms 3

A standard 3+3 dose escalation is conducted as follows:

-   -   If 0 of 3 patients experiences a DLT, escalate to next higher        dose cohort.    -   If 1 of 3 patients experiences a DLT, that cohort will be        expanded to 6 patients. If 1 of 6 patients experiences a DLT,        escalate to the next higher dose cohort;    -   If ≥2 of 3 or ≥2 of 6 patients experience a DLT, the maximum        tolerated dose (MTD) is exceeded.    -   If the MTD is exceeded, enrollment of additional patients will        be at a lower dose level. If a starting dose does not clear DLT        assessment, dose de-escalation will proceed using standard 3+3        rules

Intra-patient dose escalations are permitted for all cohorts after theintended dose level has been shown to be safe (i.e., all patientstreated at the intended dose level completed DLT assessments and ≤1patient experienced a DLT).

For dose escalation patients after 7 days and dose expansion patients,if an event meeting the definition of a DLT, but without necessarilyoccurring within the first 21 days, is observed in >33% of patients atany time, or if >33% of treated patients have withdrawn consent due totoxicity, enrollment will be held and a meeting with all Investigatorsand Sponsor will take place to review the events and discuss theirclinical significance. Based on this review, the Sponsor may elect toreduce the dose for enrolled patients and to resume enrollment of theexpansion cohort at this lower dose, or the enrollment into theexpansion cohort may be stopped.

Safety and tolerability are evaluated by means of DLTs (dose escalationcohorts only), AE reports, physical examinations, electrocardiograms andlaboratory safety evaluations.

Anti-tumor activity are assessed by the Investigator according todisease specific response criteria and described in terms of objectiveresponse rate, duration of response, progression-free survival, clinicalbenefit rate, overall survival, and disease control rate.

In Part A: Peripheral blood WBC subsets and cytokines are analyzed atbaseline and 7 days. Cytokines are additionally analyzed at 24 and 48hours.

In Parts B and C: Peripheral blood WBC subsets and cytokines areanalyzed at baseline and 7 and 21 days. Baseline and day 28 tumorbiopsies are analyzed for immune subset infiltration, as well as makersof angiogenesis, proliferation, and death.

Patients continue to receive Blautia Strain A until the patient hasconfirmed pharmacodynamics, withdraws consent, is lost to follow-up,experiences intolerable toxicity which precludes further treatment withBlautia Strain A, or treatment is discontinued at the discretion of thepatient, Investigator, or Sponsor. Patients who have objective diseaseprogression but have evidence of overall clinical benefit may, at therequest of the treating physician, continue treatment with BlautiaStrain A after discussion with the Medical Monitor.

INCORPORATION BY REFERENCE

All publications patent applications mentioned herein are herebyincorporated by reference in their entirety as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. In case of conflict, thepresent application, including any definitions herein, will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of treating cancer in a subject comprising administering tothe subject a bacterial composition comprising Blautia Strain A.
 2. Themethod of claim 1, wherein the Blautia Strain A is a strain comprisingat least 90% sequence identity to the nucleotide sequence of the Blautiamassiliensis Strain A (ATCC Deposit Number PTA-125134).
 3. (canceled) 4.The method of claim 1, wherein the Blautia Strain A is the Blautiamassiliensis Strain A (ATCC Deposit Number PTA-125134).
 5. The method ofclaim 1, wherein the cancer is selected from the group consisting ofhematological malignancy, acute nonlymphocytic leukemia, chroniclymphocytic leukemia, acute granulocytic leukemia, chronic granulocyticleukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemicleukemia, a leukocythemic leukemia, basophilic leukemia, blast cellleukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis,embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, undifferentiated cell leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma,carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidalcell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinomavillosum, carcinoma gigantocellulare, glandular carcinoma, granulosacell carcinoma, hair-matrix carcinoma, hematoid carcinoma,hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma,hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma insitu, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, carcinoma scroti, chondrosarcoma, fibrosarcoma,lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrialsarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblasticsarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma,liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoidsarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms'tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathicmultiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of Bcells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma,Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma,malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocyticsarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma,telangiectaltic sarcoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma,multiple myeloma, neuroblastoma, bladder cancer, breast cancer, ovariancancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primarymacroglobulinemia, small-cell lung tumors, primary brain tumors, stomachcancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,adrenal cortical cancer, Harding-Passey melanoma, juvenile melanoma,lentigo maligna melanoma, malignant melanoma, acral-lentiginousmelanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman'smelanoma, S91 melanoma, nodular melanoma subungal melanoma, andsuperficial spreading melanoma.
 6. The method of claim 1, wherein thecancer is prostate cancer, lung cancer, colon cancer, colorectal cancer,melanoma, breast cancer, pancreatic cancer, hepatocellular carcinoma, orlymphoma.
 7. The method of claim 1, wherein the bacterial composition isadministered orally, rectally, intravenously, intratumorally, orsubcutaneously.
 8. The method of claim 1, wherein at least 50% of thebacteria in the bacterial composition are Blautia Strain A. 9.(canceled)
 10. The method of claim 1, wherein substantially all of thebacteria in the bacterial composition are Blautia Strain A.
 11. Themethod of claim 1, wherein the bacterial composition comprises at least1×10⁶ colony forming units (CFUs) of Blautia Strain A. 12-16. (canceled)17. The method of claim 1, wherein the bacterial composition compriseslive bacteria, attenuated bacteria, or killed bacteria. 18-21.(canceled)
 22. The method of claim 1, wherein the method furthercomprises administering to the subject a second cancer therapy.
 23. Themethod of claim 22, wherein the second cancer therapy comprises theadministration of a chemotherapy agent to the subject. 24-72. (canceled)73. The method of claim 22, wherein the cancer therapy comprisesadministering an antibiotic to the subject. 74-75. (canceled)
 76. Themethod of claim 1, wherein the method further comprises administering aprebiotic to the subject.
 77. (canceled)
 78. A method of augmenting atumor environment containing an immune cell in a subject comprisingadministering a bacterial composition comprising Blautia Strain A to thesubject. 79-80. (canceled)
 81. A method of augmenting a tumorenvironment that comprises a biomarker of patient selection in a subjectcomprising administering a bacterial composition comprising BlautiaStrain A to the subject.
 82. (canceled)
 83. A method of augmenting atumor environment comprising a biomarker associated with immune cellactivity in a subject comprising administering a bacterial compositioncomprising Blautia Strain A to the subject. 84-89. (canceled)
 90. Abacterial composition comprising Blautia Strain A and a pharmaceuticallyacceptable carrier.
 91. The bacterial composition of claim 90, whereinthe Blautia Strain A is a strain comprising at least 99% sequenceidentity to the nucleotide sequence of the Blautia massiliensis Strain A(ATCC Deposit Number PTA-125134). 92-94. (canceled)
 95. The bacterialcomposition of claim 90, wherein at least 50% of the bacteria in thebacterial composition are Blautia Strain A. 96-100. (canceled)
 101. Thebacterial composition of claim 90, wherein the bacterial compositioncomprises live bacteria. 102-110. (canceled)